ExperimentMassSpectrometry
ExperimentMassSpectrometry[Samples]⟹Protocol
generates a Protocol object which can be used to determine the molecular weight of compounds by ionizing them and measuring their mass-to-charge ratio (m/z).
Mass Spectrometry (MS) is an analytical tool that measures the mass-to-charge ratio (m/z) of molecules present in a sample. The resulting data is typically presented as a mass spectrum, which is a plot of signal intensity as a function of m/z. These spectra can be used to determine the elemental or isotopic signature of a sample, identify unknown compounds within a sample, quantify known materials, and to elucidate structures of molecules. Specific applications of MS include protein identification, drug testing and discovery, food contamination detection, pesticide residue analysis, among others. Often, mass spectrometry is coupled to liquid chromatography (LC) or gas chromatography (GC) to provide additional separation and specificity, enabling the analysis of more complex samples (see also ExperimentLCMS and ExperimentGCMS). The key steps in mass spectrometry are the ionization of the analytes in the sample to create gas phase ions, followed by mass analysis or mass filtering of the ions, and finally mass detection. Two commonly used ionization techniques are electrospray ionization (ESI) and matrix-assisted laser-desorption ionization (MALDI). They are so-called 'soft' ionization methods that transfer charges to molecules with a low probability that the molecules fragment into smaller particles. With different mass analyzers, more structural details can be revealed by Tandem MS (or MS/MS). In Tandem MS, ionized samples are scanned/selected by the first mass analyzer (Quadrupole detector, MS1), before being split into smaller fragment ions in a collision cell. These fragment ions are then scanned/selected again by the second mass analyzer (Time-of-Flight or Quadrupole detector, MS2). This technique helps the sequencing of biopolymers (peptides, oligosaccharides, and oligonucleotides) by analyzing their fragments. Quantitative proteomics by multiple reaction monitoring (MRM) is also enabled by Tandem MS.
Experimental Principles
Figure 1.1: Procedural overview of a MassSpectrometry experiment. Step 1: Samples are prepared and transferred into instrument-compatible containers. Step 2: Gas phase ions are generated from (top) liquid samples by a nebulizer at high voltage source or (bottom) from solid samples by laser ablation. Step 3: Ions are guided through the mass analyzer, where they are separated according to their m/z ratio, and the mass spectrum is recorded by the detector. Step 4: The resulting patterns is analyzed. Please refer to Figure 2.1.1 through Figure 2.3.1 for the ionization and injection types that are supported by ECL, including relevant experiment options.
Instrumentation
Microflex LRF
Figure 2.1.1: Principles of a MALDI mass spectrometer. Samples or Calibrant are deposited on the MALDIPlate together with large excess of laser-adsorbing Matrix material. Sample spots are irradiated with repeated pulsed laser shots at LaserPowerRange. Laser shots repeatedly fired (ShotsPerRaster)at a specific position until a total NumberOfShots within one sample spot is reached. The laser shots trigger ablation and desorption of both sample and matrix material. Ions are generated by being protonated or deprotonated with the nearby matrix molecules. After DelayTime, AccelerationVoltage is applied to the MALDIPlate GridVoltage at a % of that to the secondary plate. This creates a potential gradient in the ionization region, a technique also called pulsed laser extraction or delayed extraction. This technique allows synchronization of ions of different kinetic energy, hus increasing resolution. Accelerated ions then travel through a set of focusing lenses at LensVoltage into the time-of-flight mass analyzer where they are separated by their mass-to-charge ratio. Ion counts within MassRange are recorded by the detector.
Xevo G2-XS QTOF
Figure 2.2.1: Principles of an electrospray quadrupole time of flight mass spectrometer (QTOF). Analytes are flowed through a capillary tube into a heated source box (SourceTemeprature) at the desired InfusionFlowRate. A confluence of the source box temperature, DesolvationTemperature, DesolvationGasFlow, and ESICapillaryVolage result in neubilization, vaporization, and ionization of the analyte stream to generate an electrospray. The electrospray enters the mass spectrometer through the source cone, which serves to focus the ions and excludes contimation via a ConeGasFlow. Within the mass spectrometer, the electrospray is futher refined via an applied voltage (DeclusteringVoltage) to yeild isolated gas-phase ions. Ions are then focused through a stepwave ion guide path via another applied voltage (StepwaveVoltage) into an ion beam. The ions travel through a quadrupole and a collision cell. Lastly, they enter a time-of-flight analyzer which resolves the ions by their mass to charge ratio according to their travel time. Spectra are continuously acquired for ions inside MassDetection hitting the detector during ScanTime until the end of RunDuration. For time independent samples, an average mass spectrum is generated. When coupled with liquid chromatography (see ExperimentLCMS) time-dependent mass spectra are generated instead.
QTRAP 6500
Figure 2.3.1: Principles of an electrospray triple quadrupole mass spectrometer (ESI-QQQ). Analytes are injected through the capillary tube into the source block at the desired InfusionFlowRate. Once the sample enters the source block, it is evaporated at the DesolvationTemperature under a stream of nitrogen gas with a pressure of DesolvationGasFlow. ESICapillaryVoltage is then applied to the capillary tubing tip to produce an electrospray, which turns into single gas-phase ions due to additional evaporation and desolvation SourceTemperature. Ions, focused by the ConeGasFlow, enter the mass spectrometer through the source cone and follow the ion path via an applied voltage (DeclusteringVoltage). They are then guided through an ion guide path via another applied voltage (IonGuideVoltange) to generate a focused ion beam. Ions travel through the first quadrupole mass analyzer (MS1), where their mass is resolved by their mass-to-charge ratio (m/z, MassDetection). After the MS1, ions enter the collision cell, where they can be fragmented by collision-induced dissociation, resulting in bond breakage and the fragmentation of the molecular ions into smaller fragments. CollisionCellExitVoltage accelerates and guides the fragmented ions to enter the second quadrupole mass analyzer (MS2), where their mass can be resolved again by their m/z (FragmentMassDetection). Spectra are continuously acquired for ions either inside MassDetection as a ranged value or as a fixed value, hitting the detector during ScanTime until the end of RunDuration, and a total mass spectrum is generated.
Experiment Options
General
IonSource
The type of ionization used to create gas phase ions from the molecules in the sample. Electrospray ionization (ESI) produces ions using an electrospray in which a high voltage is applied to a liquid to create an aerosol, and gas phase ions are formed from the fine spray of charged droplets as a result of solvent evaporation and Coulomb fission. In matrix-assisted laser desorption/ionization (MALDI), the sample is embedded in a laser energy absorbing matrix which is then irradiated with a pulsed laser, ablating and desorbing the molecules with minimal fragmentation and creating gas phase ions from the analyte molecules in the sample.
Figure 3.1: The two most commonly used ionization techniques are electrospray ionization (ESI) and matrix-assisted laser-desorption ionization (MALDI). They are both so-called 'soft' ionization methods that transfer charges to the molecules in the sample without causing their fragmentation (thus no destruction). In a MassSpectrometry experiment, use the IonSource option to indicate which ionization to use (MALDI|ESI). (TOP) In MALDI measurements, samples and calibrants are spotted onto the MALDI plate and embedded in a laser energy absorbing matrix. During the MALDI experiment, the sample is irradiated with a pulsed laser, ablating and desorbing the molecules with minimal fragmentation and creating gas phase ions from the analyte molecules in the sample. Most ions generated during MALDI measurements have a single charge, thus interpretation of acquired spectra is facilitated. MALDI is mostly used for characterization of large, thermally labile compounds, and is able to handle higher concentration of salt when compared to ESI, which is a great advantage when analyzing biological samples in situ. (BOTTOM) ESI produces ions using an electrospray in which a high voltage is applied to a liquid passing through a capillary tube to create an aerosol, and gas phase ions are formed from the fine spray of charged droplets as a result of solvent evaporation and Coulomb fission. ESI produces mostly multiply protonated ions and has broad utility for the analysis of very small and large compounds, and intact bio-molecules and protein structure.
Default Calculation: Is automatically set to ESI or MALDI, respectively, whenever ESI or MALDI specific options or IonSource are specified. If none specified, defaults to MALDI if sample contains DNA oligomers and other synthetic nucleic acid oligomers, and ESI for all other samples.
Instrument
The instrument used to perform the mass spectrometry analysis by ionization of sample and analysis by sequential mass analyzers.
Default Calculation: Is automatically set to the MALDI time-of-flight (TOF) (Model[Instrument, MassSpectrometer, "Microflex LT"]) whenever the IonSource option is set to MALDI and/or MALDI specific options are specified. Otherwise, defaults to ESI-QTOF (Model[Instrument, MassSpectrometer, "Xevo G2-XS QTOF"]).
Pattern Description: An object of type or subtype Model[Instrument, MassSpectrometer] or Object[Instrument, MassSpectrometer]
Programmatic Pattern: ObjectP[{Model[Instrument, MassSpectrometer], Object[Instrument, MassSpectrometer]}] | Automatic
MassAnalyzer
The manner of detection used to resolve and detect molecules. For now, we have 3 mass analyzers available for ExperimentMassSpectrometry. Time-Of-Flight (TOF) analyzer separates the ions by their flight time. QTOF accelerates ions through an elongated flight tube, followed by traveling down the quandrupole analyzer where only ions with selected mass to charge ratio will pass (either a mass range or specific mass value), and then passing a TOF analyzer. QQQ accelerates the ions and selects through two quadrupole analyzers. Both QTOF and QQQ have a collision cell, which optionally fragments the ions, in between the first and the second mass analyzer.
Default Calculation: For MALDI as the ion source, MassAnalyzer is automatically set to TOF detector, as TOF is the only detector that connects to MALDI for now. For ESI ion source, the detector can be TOF/QTOF for the ESI-QTOF study and quadrupole(Q)/triple-quadrupole (QQQ) for ESI-QQQ study. For APCI as the ion source, the analyzer can be quadrupole or triple-quadrupole.
InjectionType
The type of sample submission method to employ for ESI-QTOF and ESI-QQQ. In DirectInfusion, the sample is directly injected into the instrument, using either a built-in fluidics pump system (ESI-QTOF) or a syringe pump (ESI-QQQ), of the mass spectrometer without the use of any mobile phase. Due to the manual nature of the injection, this is well suited for measuring a small number of samples. Furthermore, this is recommended when high sample volumes of are available (>500 Microliter), the sample does not require cooling, and a quick result is desired. It produces a constant signal over time. FlowInjection works by injecting the sample into a flowing solvent stream using a liquid chromatography autosampler and pumps, in the absence of chromatographic separation. FlowInjection can accomodate up to 2*96 samples and is thus suited for highthrougput analyses. It produces a brief peak of signal at the time that the analyte reaches the detector.
Figure 3.2: Overview of ESI injection types. In ESI experiments, the sample can be infused into the mass spectrometer directly (DirectInfusion) or injected via an affiliated LC system (FlowInjection). In both cases, the spectra acquired over RunDuration are averaged to produce a single MassSpectrum. Use the option InjectionType to indicate which type of injection should be used in an ESI experiment at ECL (DirectInfusion|FlowInjection). (TOP) For FlowInjection, the samples (either in 2mL tubes or 96 deep well plates) are loaded into the temperature-controlled (via SampleTemperature) autosampler module of a liquid chromatography (LC) system coupled to the mass spectrometer. A small amount of sample, InjectionVolume, is then picked up and injected at a constant rate, InjectionFlowRate, into a flowing solvent stream of mobile phase (Buffer) using the HPLC pumps, similar to ExperimentHPLC / ExperimentLCMS but in the absence of chromatographic separation (no column). FlowInjection can accomodate up to 2*96 samples and is thus suited for highthrougput analyses. It produces a brief peak of signal at the time that the analyte reaches the detector. (BOTTOM) DirectInfusion is compatible with 2mL containers and 30mL reservoirs. The sample is attached to the fluidics deck of the mass spectrometer. A small amount of sample, InjectionVolume, is directly injected into the instrument at a constant rate (InjectionFlowRate) using the built-in pump system, without the use of any mobile phase. Due to the manual nature of the injection, this is well suited for measuring a small number of samples. Furthermore, this is recommended when high sample volumes of are available (>500 Microliter), the sample does not require cooling, and a quick result is desired (since no HPLC instrument and mobile phase setup is required). It produces a constant signal over time.
Default Calculation: Is automatically set to Null for MALDI mass spectrometry. If ESI mass spectrometry, is automatically set to FlowInjection if the number of samples is larger than 5, and/or the samples are inside a 96 deep well plate, and/or none of the flow injection options are set, otherwise is set to DirectInfusion.
Analytes
The compounds of interest that are present in the given samples, used to determine the other settings for the Mass Spectrometer (ex. MassDetection).
Default Calculation: If populated, will resolve to the user-specified Analytes field in the Object[Sample]. Otherwise, will resolve to the larger compounds in the sample, in the order of Proteins, Peptides, Oligomers, then other small molecules. Otherwise, set Null.
Pattern Description: List of one or more an object of type or subtype Model[Molecule], Model[Molecule, cDNA], Model[Molecule, Oligomer], Model[Molecule, Transcript], Model[Molecule, Protein], Model[Molecule, Protein, Antibody], Model[Molecule, Carbohydrate], Model[Molecule, Polymer], Model[Resin], Model[Resin, SolidPhaseSupport], Model[Lysate], Model[ProprietaryFormulation], Model[Virus], Model[Cell], Model[Cell, Mammalian], Model[Cell, Bacteria], Model[Cell, Yeast], Model[Tissue], Model[Material], or Model[Species] entries or Null.
IonMode
Default Calculation: For oligomer samples of the types Peptide, DNA, and other synthetic oligomers, is automatically set to positive ion mode. For other types of samples, defaults to positive ion mode, unless the sample is acid (Acid->True or pKa<=8).
MassDetection
Figure 3.3: In ESI-QTOF mass spectrometry experiments, MassDetection and Calibrant should be adjusted according to the type of analyte in the sample as well as the chosen ion mode. This table lists mass scan ranges and and calibrants that this experiment automatically defaults to, according to analyte types, if not user-specified: small molecules, peptides, and large molecules such as intact protein/antibodies and nucleic acid oligomers. For information about calibrants in MALDI experiments, please refer to the table listed for the Calibrant option.
Default Calculation: For MALDI-TOF measurements, is automatically set to ensure there are 3 calibrant peaks which flank the sample's molecular weight in the range. For ESI-QTOF measurements, is automatically set to one of three default mass ranges according to the molecular weight and/or the type of sample (small molecules -> 50-1200 m/z, peptides -> 350 - 2000 m/z, proteins/antibodies -> 500 - 5000 m/z). For ESI-QQQ, the mass range is set by default to be 100 - 1250 m/z if ScanMode is FullScan.
Programmatic Pattern: (RangeP[5*(Gram/Mole), 2000*(Gram/Mole)] | RangeP[2*(Gram/Mole), 100000*(Gram/Mole)] ;; RangeP[100*(Gram/Mole), 500000*(Gram/Mole)] | {RangeP[2*(Gram/Mole), 2000*(Gram/Mole)]..}) | Automatic
Calibrant
A sample with components of known mass-to-charge ratios (m/z) used to calibrate the mass spectrometer. In the chosen ion polarity mode, the calibrant should contain at least 3 masses spread over the mass range of interest.
Figure 3.4: In MALDI-TOF or ESI-QTOF mass spectrometry experiments, the Calibrant option, if not user-specified, gets defaulted according to the type of analytes inside the sample. In ESI-QQQ mass spectrometry, Calibrant, if not provided by the user, will be resolved based on IonMode.In ESI-QTOF mass spectrometry, Calibrant, if not provided by the user, is adjusted according to MassDetection - please refer to the table inside the MassDetection option for more details.
Default Calculation: Automatically set based on the sample type and MassDetection. For MALDI, is set to Model[Sample,StockSolution,Standard,"IDT ssDNA Ladder 10-60 nt, 40 ng/uL"] for DNA, and Model[Sample,StockSolution,Standard,"Peptide/Protein Calibrant Mix"] for all others.For ESI, is set to sodium iodide for peptide samples, cesium iodide for intact protein analysis. For other types of samples, is set to cesium iodide if molecular weight is above 2000 Da, to sodium iodide if molecular weight between 1200 and 2000 Da, and to sodium formate for all others (small molecule range). For ESI-QQQ, the default calibrant is polypropylene glycol (PPG) standard sample from SciEX. Where Model[Sample, "id:zGj91a71kXEO"] and Model[Sample, "id:bq9LA0JA1YJz"] are for the positive and negative ion mode, respectively.
Pattern Description: An object of type or subtype Object[Sample] or Model[Sample] or a prepared sample.
InfusionFlowRate
The flow rate at which the sample is injected into the mass spectrometer through either the DirectInfustion (the fluidics pump system (ESI-QTOF) or the syringe pump system (ESI-QQQ)) or the FlowInjection under a flowing solvent stream using a liquid chromatography autosampler). Note that source settings such as source voltage/temperature and desolvation temperature/flow rate should be adjusted according to the flow rate for improved sensitivity and spray stability.
Figure 3.5: ESI source settings should be adjusted according to the flow rate used to infuse the sample into the mass spectrometer to ensure successful spray formation and solvent desolvation. This table provides recommended settings for typical flow rate ranges for both ESI-QTOF and ESI-QQQ. Note that ESI-QTOF and ESI-QQQ take different input ranges and units. Desolvation gas flow is input as gas flow rate for ESI-QTOF and gas pressure for ESI-QQQ; ESI-QQQ has different polarity for the capillary voltage for positive and negative ion mode, while ESI-QTOF only takes positive capillary voltage as the input.
Default Calculation: In DirectInfusion: Is automatically set to 20 Microliter/Minute and 5 Microliter/Minute for ESI-QTOF and ESI-QQQ, respectively; In FlowInjection; 100 Microliter/Minute for both ESI-QQQ and ESI-QTOF; otherwise is set to Null.
Pattern Description: Greater than or equal to 1 microliter per minute and less than or equal to 600 microliters per minute or Null.
ScanTime
The duration of time allowed to pass between each spectral acquisition. Increasing this value improves sensitivity, whereas decreasing this value allows for more data points and spectra to be acquired during the RunDuration.
Default Calculation: Is automatically set to 1 second for ESI-QTOF mass spectrometry. For ESI-QQQ, in MultipleReactionMonitoring, this option is auto resolved by summing up all dwell times of each assay. For FullScan, ScanTime is set to the minimum allowed for the specified MassDetection range (3 microseconds per discrete point in the MassDetection range). For the rest of scan modes, is set to 5 Millisecond (0.005 Second) by default. Otherwise is set to Null.
Pattern Description: Greater than or equal to 0.005 milliseconds and less than or equal to 10 seconds or Null.
NumberOfReplicates
Number of times each of the input samples should be analyzed using identical experimental parameters.
MALDI Ionization
NumberOfShots
The total number of times the laser is fired by the MALDI mass spectrometer during data acquisition.
Default Calculation: Is automatically set to 500 for MALDI mass spectrometry, otherwise is set to Null.
ShotsPerRaster
The number of repeated laser shots made between each raster movement within a well during a MALDI measurement.
Default Calculation: Is automatically set to 12 for MALDI mass spectrometry, otherwise is set to Null.
LaserPowerRange
The min and max laser power used during analysis (given as a relative percentage of the mass spectrometer's actual laser power). Adjust this value to increase resolution and reduce noise.
Default Calculation: Automatically set according to sample type and molecular weight of the analyte of interest. For DNA below 3500 Da, this is set to 55-75% and for DNA above 3500 Da to 65-85%. For all other samples it is set to 25-75% if below 6000 Da and 25-90% otherwise.
Pattern Description: A span from anything greater than or equal to 0 percent and less than or equal to 100 percent in increments of 1 percent to anything greater than or equal to 0 percent and less than or equal to 100 percent in increments of 1 percent or Null.
Programmatic Pattern: (RangeP[0*Percent, 100*Percent, 1*Percent] ;; RangeP[0*Percent, 100*Percent, 1*Percent] | Automatic) | Null
DelayTime
The duration of time that is allowed to pass between the laser pulse and the application of the acceleration voltage in order to control for differences in kinetic energy introduced during the ionization process. Use a longer delay time for samples with a higher molecular weight.
Default Calculation: A delay of 250 ns is used for DNA oligomers. A 150 ns delay is used for all other samples.
Pattern Description: Greater than or equal to 0 nanoseconds and less than or equal to 5000 nanoseconds or Null.
CalibrantLaserPowerRange
The min and max laser power used during calibration (given as a relative percentage of the mass spectrometer's actual laser power).
Default Calculation: Is automatically set to use the same laser power range determined for the analyte.
Pattern Description: A span from anything greater than or equal to 0 percent and less than or equal to 100 percent in increments of 1 percent to anything greater than or equal to 0 percent and less than or equal to 100 percent in increments of 1 percent or Null.
Programmatic Pattern: (RangeP[0*Percent, 100*Percent, 1*Percent] ;; RangeP[0*Percent, 100*Percent, 1*Percent] | Automatic) | Null
CalibrantNumberOfShots
Default Calculation: Is automatically set to 100 for MALDI mass spectrometry, otherwise is set to Null.
MALDI Sample Preparation
SpottingPattern
Indicates if MALDI plate wells are skipped during spotting to decrease any cross contamination risks; All indicates every well on the plate will be spotted and Spaced indicates every other well is filled.
Default Calculation: Is automatically set to All for MALDI mass spectrometry, otherwise is set to Null.
SpottingDryTime
The minimum amount of time the samples are left to dry after they have been aliquoted onto the MALDI plate.
Default Calculation: Is automatically set to 15 minutes for MALDI mass spectrometry, otherwise is set to Null.
MALDIPlate
The plate spotted with samples and calibrants mixed with laser energy absorbing matrix material. The MALDI plate is subsequenctly placed into the mass spectrometer's reduced pressure sample chamber and irradiated with laser pulses to produce gas phase ions that enter the mass spectrometer for mass analysis.
Default Calculation: Is automatically set to Model[Container, Plate, MALDI, "96-well Ground Steel MALDI Plate"] for MALDI mass spectrometry, otherwise is set to Null.
Pattern Description: An object of type or subtype Model[Container, Plate], Object[Container, Plate], Model[Container, Plate, MALDI], or Object[Container, Plate, MALDI] or a prepared sample or Null.
Programmatic Pattern: ((ObjectP[{Model[Container, Plate], Object[Container, Plate], Model[Container, Plate, MALDI], Object[Container, Plate, MALDI]}] | _String) | Automatic) | Null
Matrix
The laser-absorbing reagent co-spotted with the input samples in order to assist ionization of the sample.
Default Calculation: Automatically set according to sample type and ion mode. For DNA oligomers and positive ion mode, HPA MALDI matrix is used. Otherwise, the PreferredMALDIMatrix of the calibrant used for this sample is used, and if that is not informed, it defaults to THAP MALDI matrix.
Pattern Description: An object of type or subtype Model[Sample, Matrix] or Object[Sample] or a prepared sample or Null.
Programmatic Pattern: ((ObjectP[{Model[Sample, Matrix], Object[Sample]}] | _String) | Automatic) | Null
CalibrantMatrix
The laser-absorbing reagent co-spotted with the input calibrants in order to assist ionization of the sample.
Default Calculation: Automatically set to according to the PreferredMatrix field of the calibrant models, else will be resolved to be the same as Matrix.
Pattern Description: An object of type or subtype Model[Sample, Matrix] or Object[Sample] or a prepared sample or Null.
Programmatic Pattern: ((ObjectP[{Model[Sample, Matrix], Object[Sample]}] | _String) | Automatic) | Null
SpottingMethod
Indicates if or how to layer the input samples and matrix onto the MALDI plate in order to form sample and calibration spots for analysis.
SampleVolume
Default Calculation: Is automatically set to 0.8 Microliter for MALDI mass spectrometry, otherwise is set to Null.
Pattern Description: Greater than or equal to 0.5 microliters and less than or equal to 2 microliters or Null.
CalibrantVolume
Default Calculation: Is automatically set to 0.8 Microliter for MALDI mass spectrometry, otherwise is set to Null.
Pattern Description: Greater than or equal to 0.5 microliters and less than or equal to 2 microliters or Null.
MatrixControlScans
Indicates if matrix control samples will be spotted to MALDI plate and investigated as the blank control during the experiment for each different combination of MALDI experiment parameters.
Default Calculation: Is automatically set to True for MALDI mass spectrometry, otherwise is set to Null.
ESI Flow Injection
SampleTemperature
The temperature at which the samples are kept in the instrument's autosampler prior to injection. This option can only be changed for ESI-QQQ and ESI-QTOF using FlowInjection as the InjectionType.
Default Calculation: Is automatically set to Ambient for FlowInjection ESI mass spectrometry, otherwise is set to Null.
Pattern Description: Ambient or greater than or equal to 5 degrees Celsius and less than or equal to 40 degrees Celsius or Null.
Buffer
The solvent pumped through the flow path, carrying the injected sample to the ionization source where the analytes are ionized via electrospray ionization.
Default Calculation: Is automatically set to Model[Sample,StockSolution,"0.1% FA with 5% Acetonitrile in Water, LCMS-grade"] for FlowInjection ESI mass spectrometry, otherwise is set to Null.
Pattern Description: An object of type or subtype Object[Sample] or Model[Sample] or a prepared sample or Null.
NeedleWashSolution
Default Calculation: Is automatically set to Model[Sample,StockSolution,"20% Methanol in MilliQ Water"] for FlowInjection ESI mass spectrometry, otherwise is set to Null.
Pattern Description: An object of type or subtype Object[Sample] or Model[Sample] or a prepared sample or Null.
InjectionVolume
Default Calculation: Is automatically set to 10 Microliter for FlowInjection ESI mass spectrometry (For Both QQQ and QTOF), otherwise is set to Null.
Pattern Description: Greater than or equal to 1 microliter and less than or equal to 50 microliters or Null.
ESI-QQQ Direct Infusion
InfusionVolume
The physical quantity of sample loaded into the syringe (for now ESI-QQQ only), when using syringe pump to infusion load the sample.
Default Calculation: Is automatically set to 500 Microliter for DirectInfusion ESI mass spectrometry, otherwise is set to Null.
Pattern Description: Greater than or equal to 0.05 milliliters and less than or equal to 10 milliliters or Null.
InfusionSyringe
Default Calculation: For ESI-QQQ, is automatically resolved based on the infusion volume: (0.01 mL ~ 0.99 mL) -> Model[Container, Syringe, "1mL All-Plastic Disposable Syringe"]; (1.01 mL ~ 2.99 mL) ->Model[Container, Syringe, "3mL Sterile Disposable Syringe"];(3.00 mL,4.99 mL)->Model[Container, Syringe, "5mL Sterile Disposable Syringe"];(5.0mL,9.9mL)->Model[Container, Syringe, "10mL Syringe"]. Otherwise is set to Null.
Pattern Description: An object of type or subtype Model[Container, Syringe] or Object[Container, Syringe] or a prepared sample or Null.
Programmatic Pattern: ((ObjectP[{Model[Container, Syringe], Object[Container, Syringe]}] | _String) | Automatic) | Null
Method Information
RunDuration
Default Calculation: Is automatically set to 1 minute for ESI-QTOF and ESI-QQQ mass spectrometry, otherwise is set to Null.
Pattern Description: Greater than or equal to 0.1 minutes and less than or equal to 60 minutes or Null.
ESI Ionization
ESICapillaryVoltage
This option (also known as Ion Spray Voltage) indicates the absolute voltage applied to the tip of the stainless steel capillary tubing in order to produce charged droplets. Adjust this voltage to maximize sensitivity. For ESI-QTOF, most compounds are optimized between 0.5 and 3.2 kV in ESI positive ion mode and 0.5 and 2.6 kV in ESI negative ion mode. For ESI-QQQ this parameter is 5.5 kV for positive ion mode and -4.5 kV for negative ion mode, for standard flow UPLC a value of 0.5 kV is typically best for maximum sensitivity. Thie parameter can be altered according to sample type. For low flow applications, best sensitivity will be achieved with a relatively high value in ESI positive (e.g. 3.0 kV for ESI-QTOF and 5.5 or -4.5 kV for ESI-QQQ positive and negative, respectively).
Default Calculation: For ESI-QTOF: Is automatically set according to the flow rate: For ESI-QTOF: 0-0.02 ml/min -> 3.0 kV (Positive) or 2.8 kV (Negative), 0.021-0.1 ml/min -> 2.5 kV, 0.101 - 0.3 ml/min -> 2.0 kV, 0.301 - 0.5 ml/min -> 1.5 kV, > 0.5 ml/min -> 1.0 kv . For ESI-QQQ: < 0.02 ml/min -> 5.5 kV or -4.5 kV, 0.02-0.1 ml/min -> 4.5 kV or -4.0 KV, >0.1 ml/min -> 4.0 kV and -4.0 kV, for positive and negative IonMode respectively.
Pattern Description: Greater than or equal to -4.5 kilovolts and less than or equal to 5.5 kilovolts or Null.
DeclusteringVoltage
For ESI-QTOF, indicates the voltage between the ion block (the reduced-pressure chamber of the source block) and the stepwave ion guide (the optics before the quadrupole mass analyzer). This voltage attracts charged ions from the capillary tip into the ion block leading into the mass spectrometer. For ESI-QTOF, this voltage is typically set to 25-100 V and its tuning has little effect on sensitivity. For ESI-QQQ, this controls the voltage applied between the orifice (where ions enter the mass spectrometer) and the ion guide to prevent the ionized small particles from aggregating together. It's normally optimized between -300 to 300 V, and its sensitivity depends on chemical composition and charge state.
Default Calculation: Is automatically set to any specified method; otherwise, for ESI-QTOF, is set to 40 Volt; and for ESI-QQQ, is set to 90 Volt and -90 respectively for positive and negative ion mode.
Pattern Description: Greater than or equal to -400 volts and less than or equal to 400 volts or Null.
StepwaveVoltage
This is a unique option for ESI-QTOF. It indicates the voltage offset between the 1st and 2nd stage of the ion guide which leads ions coming from the sample cone towards the quadrupole mass analyzer. This voltage normally optimizes between 25 and 150 V and should be adjusted for sensitivity depending on compound and charge state. For multiply charged species it is typically set to to 40-50 V, and higher for singly charged species. In general higher cone voltages (120-150 V) are needed for larger mass ions such as intact proteins and monoclonal antibodies. It also has greatest effect on in-source fragmentation and should be decreased if in-source fragmentation is observed but not desired.
Default Calculation: Is automatically set according to the sample type (proteins, antibodies and analytes with MW > 2000 -> 120 V, DNA and synthetic nucleic acid oligomers -> 100 V, all others (including peptides and small molecules) -> 40 V).
Pattern Description: Greater than or equal to 0.1 volts and less than or equal to 200 volts or Null.
SourceTemperature
The temperature setting of the source block. Heating the source block discourages condensation and decreases solvent clustering in the reduced vacuum region of the source. For ESI-QTOF, This temperature setting is flow rate and sample dependent. Typical values are between 60 to 120 Celsius. For thermally labile analytes, a lower temperature setting is recommended. For ESI-QQQ, this values is set to 150 Celsius cannot be changed.
Default Calculation: For ESI-QTOF: is automatically set according to the flow rate (0-0.02 ml/min -> 100 Celsius, 0.021-0.3 ml/min -> 120 Celsius, >0.3 ml/min -> 150 Celsius). For ESI-QQQ, this value is locked to be 150 Celsius.
Pattern Description: Greater than or equal to 25 degrees Celsius and less than or equal to 150 degrees Celsius or Null.
DesolvationTemperature
The temperature setting for the ESI desolvation heater that controls the nitrogen gas temperature used for solvent evaporation to produce single gas phase ions from the ion spray. Similarly to DesolvationGasFlow, this setting is dependant on solvent flow rate and composition. A typical range is from 150 to 650 Celsius for ESI-QTOF and 350 to 600 Celsius for ESI-QQQ.
Default Calculation: Is automatically set according to the flow rate: For ESI-QTOF (0-0.02 ml/min -> 200 Celsius, 0.021-0.1 ml/min -> 350 Celsius, 0.101-0.3 -> 450 Celsius, 0.301->0.5 ml/min -> 500 Celsius, >0.500 ml/min -> 600 Celsius); For ESI-QQQ (0-0.02 ml/min -> 350 Celsius, 0.021-0.1 ml/min -> 400 Celsius, 0.101-0.3 -> 450 Celsius, 0.301->0.5 ml/min -> 500 Celsius, >0.500 ml/min -> 600 Celsius); .
Pattern Description: Greater than or equal to 0 degrees Celsius and less than or equal to 750 degrees Celsius or Null.
DesolvationGasFlow
The nitrogen gas flowing around the electrospray inlet capillary in order to desolvate and nebulize analytes. Similarly to DesolvationTemperature, the ideal setting is dependent on solvent flow rate and composition. When MassAnalyzer is QQQ, this value is in terms of pressure (PSI). When MassAnalyzer is QTOF, this value is in terms of flow rate (Liter/Hour).
Default Calculation: Is automatically set according to the flow rate: for ESI-QTOF (0-0.02 ml/min -> 600 L/H, 0.021-0.3 ml/min -> 800 L/H, 0.301-0.5 -> 1000 L/H, >0.500 ml/min -> 1200 L/H); and (0-0.02 ml/min -> 20 PSI, 0.02-0.3 ml/min -> 40 PSI, 0.301-0.500 ml/min -> 60 PSI, >0.500 ml/min -> 80) for ESI-QQQ.
Pattern Description: Greater than or equal to 55 liters per hour and less than or equal to 1200 liters per hour or greater than or equal to 0 pounds‐force per inch squared and less than or equal to 85 pounds‐force per inch squared or Null.
Programmatic Pattern: ((RangeP[55*(Liter/Hour), 1200*(Liter/Hour)] | RangeP[0*PSI, 85*PSI]) | Automatic) | Null
ConeGasFlow
The nitrogen gas flow ejected around the sample inlet cone (the spherical metal plate acting as a first gate between the sprayer and the reduced pressure chamber, the ion block). This gas flow is used to minimize the formation of solvent ion clusters. It also helps reduce adduct ions and directing the spray into the ion block while keeping the sample cone clean. The same parameter is referred to as Curtain Gas Pressure for ESI-QQQ. Typical values are between 0 and 150 L/h for ESI-QTOF or 20 to 55 PSI for ESI-QQQ.
Default Calculation: Is automatically set to 50 Liter/Hour for ESI-QTOF and 50 PSI for ESI-QQQ, and is set to Null in MALDI-TOF. Is not recommended to set to a smaller value of 40 PSI in ESI-QQQ, due to potential deposition of the sample inside the instrument that will lead to contamination.
Pattern Description: Greater than or equal to 0 liters per hour and less than or equal to 300 liters per hour or greater than or equal to 20 pounds‐force per inch squared and less than or equal to 55 pounds‐force per inch squared or Null.
Programmatic Pattern: ((RangeP[0*(Liter/Hour), 300*(Liter/Hour)] | RangeP[20*PSI, 55*PSI]) | Automatic) | Null
IonGuideVoltage
This option (also known as Entrance Potential (EP)) is a unique option of ESI-QQQ. This parameter indicates electric potential applied to the Ion Guide in ESI-QQQ, which guides and focuses the ions through the high-pressure ion guide region.
Default Calculation: Is automatically set to 10 V for positive ions, or –10 V for negative ions in ESI-QQQ, and can be changed between 2-15 V in both positive and negative mode. This value is set to Null in ESI-QTOF.
Pattern Description: Greater than or equal to -15 volts and less than or equal to -2 volts or greater than or equal to 2 volts and less than or equal to 15 volts or Null.
MALDI Mass Analysis
AccelerationVoltage
The voltage applied to the MALDI plate in order to accelerate ions towards the detector. Increase this voltage to increase the sensitivity or decrease it to increase the resolution.
Default Calculation: Automatically set to 19.5 kV for MALDI mass spectrometry, otherwise set to Null.
Pattern Description: Greater than or equal to 0.1 kilovolts and less than or equal to 20 kilovolts or Null.
GridVoltage
The voltage applied to a secondary plate above the MALDI plate in order to create a gradient such that ions with lower initial kinetic energies are accelerated faster than samples with higher kinetic energies which have drifted farther from the MALDI plate. Use a lower grid voltage for samples with a higher molecular weight.
Default Calculation: Automatically set to 18.15 kV for MALDI mass spectrometry, otherwise set to Null.
Pattern Description: Greater than or equal to 0.1 kilovolts and less than or equal to 20 kilovolts or Null.
LensVoltage
The voltage applied to the electrostatic ion focusing lens located at the entrance of the mass analyser.
Default Calculation: Automatically set to 7.8 kV for MALDI mass spectrometry, otherwise set to Null.
Pattern Description: Greater than or equal to 0.05 kilovolts and less than or equal to 10 kilovolts or Null.
Gain
The signal amplification factor applied to the detector in MALDI-TOF analysis. A gain factor of one corresponds to the lowest voltage applied to the electron multiplier. Larger values can increase signal intensity, but may cause saturation and decreased signal to noise.
Tandem Mass Spectrometry
ScanMode
The acquisition and selection sequence when MassAnalyzer is TripleQuadrupole. Different scan modes will apply selections to the first (MS1) and third (MS2) quadrupole at specific times. In Full Scan Mode, the entire MS1 range is scanned and fragmentation is off. In SelectedIonMonitoring, select MS1 masses (per the MassDetection option) are monitored and measured without fragmentation. In PrecursorIonScan mode, fragmentation is on, and fragment ion is selected (per the FragmentMassDetection option), while MS1 masses are scanned across a range (MassDetection). In NeutralIonLoss mode, both MS1 and MS2 masses are scanned, in order to track specific MS1/MS2 combinations for neutral ion loss. In ProductIonScan, an MS1 mass is selected (MassDetection) and a range of MS2 mass is scanned in order to survey fragmentation patterns of the parent mass. In MultipleReactionMonitoring mode, both MS1 and MS2 are selected with specific intact and fragment ion pairs are monitored. ScanMode is automatically set to FullScan (no fragmentation of the sample, collecting full mass range scan).
Figure 3.6: Various mass spectrometry scan modes available for ESI-QQQ. In Full Scan Mode, the entire MS1 range is scanned and fragmentation is off. In SelectedIonMonitoring, select MS1 masses (per the MassDetection option) are monitored and measured without fragmentation. In PrecursorIonScan mode, fragmentation is on, and fragment ion is selected (per the FragmentMassDetection option), while MS1 masses are scanned across a range (MassDetection). In NeutralIonLoss mode, both MS1 and MS2 masses are scanned, to track specific MS1/MS2 combinations for neutral ion loss. In ProductIonScan, an MS1 mass is selected (MassDetection) and a range of MS2 mass is scanned to survey fragmentation patterns of the parent mass. In MultipleReactionMonitoring mode, both MS1 and MS2 are selected - specific intact and fragment ion pairs are monitored.
Default Calculation: For ESI-QQQ this value will be automatically resolved based-on tandem mass related input. For ESI-QTOF and MALDI-TOF, this will be resolved to Null.
Pattern Description: FullScan, SelectedIonMonitoring, PrecursorIonScan, NeutralIonLoss, ProductIonScan, or MultipleReactionMonitoring or Null.
FragmentMassDetection
The mass scan range for the second mass analyzer (MS2) in the Tandem MS study. The second mass anaylzer screens and scans the ion after the incoming ions have been fragmented by collision cells. This option can be set at one specific mass value (mass selection mode), or at a mass range (mass scan mode). This option will be checked to match the corresponding ScanMode.
Default Calculation: Is automatically resolved as Null for FullScan and SelectedIonMonitoring mode. Is set to 5 - 1250 m/z by default for ProductionIonScan modes and resolved to be the same as MassDetection in NeutralIonLoss model, and resolved based on MassDetection or 500 m/z for PrecursorIonScan and MultipleReactionMonitoring mode.
Programmatic Pattern: ((RangeP[5*(Gram/Mole), 2000*(Gram/Mole)] | RangeP[5*(Gram/Mole), 2000*(Gram/Mole)] ;; RangeP[5*(Gram/Mole), 2000*(Gram/Mole)] | {RangeP[5*(Gram/Mole), 2000*(Gram/Mole)]..}) | Automatic) | Null
MassTolerance
This options indicates the step size of both MS1 and MS2 when both or either one of them are set in mass selection mode. This value indicates the mass range used to find a peak with twice the entered range. For example, for a mass range 100 Da to 200 Da and step size 0.1, the instrument scans 99.95 to 100.05 (records as value 100), 100.05 to 101.15 (records as value 101) and 199.95 to 200.05 (records as value 200).
Default Calculation: This option will be set to Null if using MALDI-TOF and ESI-QTOF. For ESI-QQQ, if both of the mass anaylzer are in mass selection mode (SelectedIonMonitoring and MultipleReactionMonitoring mode), this option will be auto resolved to Null. In all other mass scan modes in ESI-QQQ, this option will be automatically resolved to 0.1 g/mol.
Pattern Description: Greater than or equal to 0.01 grams per mole and less than or equal to 1 gram per mole or Null.
Fragment
Determines whether to have ions dissociate upon collision with neutral gas species and to measure the resulting product ions. Also known as tandem mass spectrometry or MS/MS (as opposed to MS).
Default Calculation: For ESI-QQQ, this option will be automatically resolved by the corresponding scan modes (False for FullScan and SelectedIonMonitoring Method, True for PrecursorIonScan, NeutralIonLoss, ProductIonScan and MultipleReactionMonitoring mode). For ESI-QTOF and MALDI-TOF this options will be resolved to False.
CollisionEnergy
The potential used in the collision cell to fragment the incoming ions. Changing the collision energy will change the fragmentation pattern of the incoming ion. High collision energy gives higher ion intensities but the mass patterns will also be more complex. Low collision energy gives simpler mass patterns with lower intensity.
Default Calculation: Is automatically set to 30 V (Positive) and -30 V (Negative) if the collision option is True. This value is Null when Collision option is False and when scan mode is set to MultipleReactionMonitoring.
Pattern Description: Greater than or equal to 5 volts and less than or equal to 180 volts or greater than or equal to -180 volts and less than or equal to 5 volts or list of one or more greater than or equal to 5 volts and less than or equal to 180 volts or greater than or equal to -180 volts and less than or equal to 5 volts entries or Null.
Programmatic Pattern: (((RangeP[5*Volt, 180*Volt] | RangeP[-180*Volt, 5*Volt]) | {(RangeP[5*Volt, 180*Volt] | RangeP[-180*Volt, 5*Volt])..}) | Automatic) | Null
CollisionCellExitVoltage
Also known as the Collision Cell Exit Potential (CXP). This value focuses and accelerates the ions out of collision cell (Q2) and into 2nd mass analyzer (MS 2). This potential is tuned to ensure successful ion acceleration out of collision cell and into MS2, and can be adjusted to reach the maximal signal intensity. This option is unique to ESI-QQQ for now, and only required when Fragment ->True and/or in ScanMode that achieves tandem mass feature (PrecursorIonScan, NeutralIonLoss,ProductIonScan,MultipleReactionMonitoring). For non-tandem mass ScanMode (FullScan and SelectedIonMonitoring) and other massspectrometer (ESI-QTOF and MALDI-TOF), this option is resolved to Null.
Default Calculation: Is automatically set to 15 V (Positive mode) or -15 V (Negative mode) if the collision option is True and using QQQ as the mass analyzer.
DwellTime
The duration of time for which spectra are acquired at the specific mass detection value for SelectedIonMonitoring and MultipleReactionMonitoring mode in ESI-QQQ.
Default Calculation: Is automatically set to 200 microsecond for ESI-QQQ mass spectrometry at SelectedIonMonitoring mode, otherwise is set to Null.
Pattern Description: Greater than or equal to 5 milliseconds and less than or equal to 2000 milliseconds or list of one or more greater than or equal to 5 milliseconds and less than or equal to 2000 milliseconds entries or Null.
Programmatic Pattern: ((RangeP[5*Millisecond, 2000*Millisecond] | {RangeP[5*Millisecond, 2000*Millisecond]..}) | Automatic) | Null
NeutralLoss
A neutral loss scan is performed on ESI-QQQ instrument by scanning the sample through the first quadrupole (Q1). The ions are then fragmented in the collision cell. The second mass analyzer is then scanned with a fixed offset to MS1. This option represents the value of this offset.
MultipleReactionMonitoringAssays
In ESI-QQQ, the ion corresponding to the compound of interest is targetted with subsequent fragmentation of that target ion to produce a range of daughter ions. One (or more) of these fragment daughter ions can be selected for quantitation purposes. Only compounds that meet both these criteria, i.e. specific parent ion and specific daughter ions corresponding to the mass of the molecule of interest are detected within the mass spectrometer. The mass assays (MS1/MS2 mass value combinations) for each scan, along with the CollisionEnergy and DwellTime (length of time of each scan).
Default Calculation: Need to fill in order to use Multiple Reaction Monitoring mode. Will auto switch to Selected Reaction Monitoring mode if only one mass assay/dwell time is input. Default dwell time is 200 micro second.
Pattern Description: List of one or more {Mass Selection Values, CollisionEnergies, Fragment Mass Selection Values, Dwell Times} entries or Null.
Programmatic Pattern: ({{GreaterP[0*(Gram/Mole)], (RangeP[5*Volt, 180*Volt] | RangeP[-180*Volt, 5*Volt]) | Automatic, GreaterP[0*(Gram/Mole)], GreaterP[0*Second] | Automatic}..} | Automatic) | Null
Post Experiment
CalibrantStorageCondition
For each calibrants used in the experiment, the non-default condition under which the calibrants should be stored after the protocol is complete. If not set, the storage condition will be stored according to their current StorageCondition.
Pattern Description: {AmbientStorage, Refrigerator, Freezer, DeepFreezer, CryogenicStorage, YeastIncubation, YeastShakingIncubation, BacterialIncubation, BacterialShakingIncubation, MammalianIncubation, ViralIncubation, CrystalIncubation, AcceleratedTesting, IntermediateTesting, LongTermTesting, UVVisLightTesting} or Disposal or Null.
SamplesInStorageCondition
The non-default conditions under which the SamplesIn of this experiment should be stored after the protocol is completed. If left unset, SamplesIn will be stored according to their current StorageCondition.
Pattern Description: {AmbientStorage, Refrigerator, Freezer, DeepFreezer, CryogenicStorage, YeastIncubation, YeastShakingIncubation, BacterialIncubation, BacterialShakingIncubation, MammalianIncubation, ViralIncubation, CrystalIncubation, AcceleratedTesting, IntermediateTesting, LongTermTesting, UVVisLightTesting} or Disposal or Null.
Sample Prep Options
Sample Preparation
PreparatoryUnitOperations
Specifies a sequence of transferring, aliquoting, consolidating, or mixing of new or existing samples before the main experiment. These prepared samples can be used in the main experiment by referencing their defined name. For more information, please reference the documentation for ExperimentSampleManipulation.
Pattern Description: List of one or more unit Operation ManualSamplePreparation or RoboticSamplePreparation or unit Operation must match SamplePreparationP entries or Null.
Programmatic Pattern: {((ManualSamplePreparationMethodP | RoboticSamplePreparationMethodP) | SamplePreparationP)..} | Null
PreparatoryPrimitives
Specifies a sequence of transferring, aliquoting, consolidating, or mixing of new or existing samples before the main experiment. These prepared samples can be used in the main experiment by referencing their defined name. For more information, please reference the documentation for ExperimentSampleManipulation.
Pattern Description: List of one or more a primitive with head Define, Transfer, Mix, Aliquot, Consolidation, FillToVolume, Incubate, Filter, Wait, Centrifuge, or Resuspend entries or Null.
Preparatory Incubation
Incubate
Indicates if the SamplesIn should be incubated at a fixed temperature prior to starting the experiment or any aliquoting. Sample Preparation occurs in the order of Incubation, Centrifugation, Filtration, and then Aliquoting (if specified).
Default Calculation: Resolves to True if any of the corresponding Incubation options are set. Otherwise, resolves to False.
IncubationTemperature
Temperature at which the SamplesIn should be incubated for the duration of the IncubationTime prior to starting the experiment.
Pattern Description: Ambient or greater than or equal to -20 degrees Celsius and less than or equal to 500 degrees Celsius or Null.
Programmatic Pattern: ((Ambient | RangeP[$MinIncubationTemperature, $MaxIncubationTemperature]) | Automatic) | Null
IncubationTime
Duration for which SamplesIn should be incubated at the IncubationTemperature, prior to starting the experiment.
Mix
Default Calculation: Automatically resolves to True if any Mix related options are set. Otherwise, resolves to False.
MixType
Default Calculation: Automatically resolves based on the container of the sample and the Mix option.
Pattern Description: Roll, Vortex, Sonicate, Pipette, Invert, Stir, Shake, Homogenize, Swirl, Disrupt, or Nutate or Null.
MixUntilDissolved
Indicates if the mix should be continued up to the MaxIncubationTime or MaxNumberOfMixes (chosen according to the mix Type), in an attempt dissolve any solute. Any mixing/incubation will occur prior to starting the experiment.
Default Calculation: Automatically resolves to True if MaxIncubationTime or MaxNumberOfMixes is set.
MaxIncubationTime
Maximum duration of time for which the samples will be mixed while incubated in an attempt to dissolve any solute, if the MixUntilDissolved option is chosen. This occurs prior to starting the experiment.
Default Calculation: Automatically resolves based on MixType, MixUntilDissolved, and the container of the given sample.
IncubationInstrument
Default Calculation: Automatically resolves based on the options Mix, Temperature, MixType and container of the sample.
Pattern Description: An object of type or subtype Model[Instrument, Roller], Model[Instrument, OverheadStirrer], Model[Instrument, Vortex], Model[Instrument, Shaker], Model[Instrument, BottleRoller], Model[Instrument, Roller], Model[Instrument, Sonicator], Model[Instrument, HeatBlock], Model[Instrument, Homogenizer], Model[Instrument, Disruptor], Model[Instrument, Nutator], Model[Instrument, Thermocycler], Model[Instrument, EnvironmentalChamber], Model[Instrument, Pipette], Object[Instrument, Roller], Object[Instrument, OverheadStirrer], Object[Instrument, Vortex], Object[Instrument, Shaker], Object[Instrument, BottleRoller], Object[Instrument, Roller], Object[Instrument, Sonicator], Object[Instrument, HeatBlock], Object[Instrument, Homogenizer], Object[Instrument, Disruptor], Object[Instrument, Nutator], Object[Instrument, Thermocycler], Object[Instrument, EnvironmentalChamber], or Object[Instrument, Pipette] or Null.
AnnealingTime
Minimum duration for which the SamplesIn should remain in the incubator allowing the system to settle to room temperature after the IncubationTime has passed but prior to starting the experiment.
IncubateAliquotContainer
The desired type of container that should be used to prepare and house the incubation samples which should be used in lieu of the SamplesIn for the experiment.
Programmatic Pattern: ((ObjectP[Model[Container]] | {GreaterEqualP[1, 1] | (Automatic | Null), (ObjectP[{Model[Container], Object[Container]}] | _String) | Automatic}) | Automatic) | Null
IncubateAliquotDestinationWell
The desired position in the corresponding IncubateAliquotContainer in which the aliquot samples will be placed.
Default Calculation: Automatically resolves to A1 in containers with only one position. For plates, fills wells in the order provided by the function AllWells.
IncubateAliquot
The amount of each sample that should be transferred from the SamplesIn into the IncubateAliquotContainer when performing an aliquot before incubation.
Default Calculation: Automatically set as the smaller between the current sample volume and the maximum volume of the destination container.
Pattern Description: All or greater than or equal to 1 microliter and less than or equal to 20 liters or Null.
Preparatory Centrifugation
Centrifuge
Indicates if the SamplesIn should be centrifuged prior to starting the experiment or any aliquoting. Sample Preparation occurs in the order of Incubation, Centrifugation, Filtration, and then Aliquoting (if specified).
Default Calculation: Resolves to True if any of the corresponding Centrifuge options are set. Otherwise, resolves to False.
CentrifugeInstrument
Pattern Description: An object of type or subtype Model[Instrument, Centrifuge] or Object[Instrument, Centrifuge] or Null.
Programmatic Pattern: (ObjectP[{Model[Instrument, Centrifuge], Object[Instrument, Centrifuge]}] | Automatic) | Null
CentrifugeIntensity
The rotational speed or the force that will be applied to the samples by centrifugation prior to starting the experiment.
Pattern Description: Greater than 0 revolutions per minute or greater than 0 standard accelerations due to gravity on the surface of the earth or Null.
Programmatic Pattern: ((GreaterP[0*RPM] | GreaterP[0*GravitationalAcceleration]) | Automatic) | Null
CentrifugeTime
CentrifugeTemperature
The temperature at which the centrifuge chamber should be held while the samples are being centrifuged prior to starting the experiment.
Pattern Description: Ambient or greater than or equal to -10 degrees Celsius and less than or equal to 40 degrees Celsius or Null.
CentrifugeAliquotContainer
The desired type of container that should be used to prepare and house the centrifuge samples which should be used in lieu of the SamplesIn for the experiment.
Programmatic Pattern: ((ObjectP[Model[Container]] | {GreaterEqualP[1, 1] | (Automatic | Null), (ObjectP[{Model[Container], Object[Container]}] | _String) | Automatic}) | Automatic) | Null
CentrifugeAliquotDestinationWell
The desired position in the corresponding AliquotContainer in which the aliquot samples will be placed.
Default Calculation: Automatically resolves to A1 in containers with only one position. For plates, fills wells in the order provided by the function AllWells.
CentrifugeAliquot
The amount of each sample that should be transferred from the SamplesIn into the CentrifugeAliquotContainer when performing an aliquot before centrifugation.
Default Calculation: Automatically set as the smaller between the current sample volume and the maximum volume of the destination container.
Pattern Description: All or greater than or equal to 1 microliter and less than or equal to 20 liters or Null.
Preparatory Filtering
Filtration
Indicates if the SamplesIn should be filter prior to starting the experiment or any aliquoting. Sample Preparation occurs in the order of Incubation, Centrifugation, Filtration, and then Aliquoting (if specified).
Default Calculation: Resolves to True if any of the corresponding Filter options are set. Otherwise, resolves to False.
FiltrationType
Default Calculation: Will automatically resolve to a filtration type appropriate for the volume of sample being filtered.
FilterInstrument
Default Calculation: Will automatically resolved to an instrument appropriate for the filtration type.
Pattern Description: An object of type or subtype Model[Instrument, FilterBlock], Object[Instrument, FilterBlock], Model[Instrument, PeristalticPump], Object[Instrument, PeristalticPump], Model[Instrument, VacuumPump], Object[Instrument, VacuumPump], Model[Instrument, Centrifuge], Object[Instrument, Centrifuge], Model[Instrument, SyringePump], or Object[Instrument, SyringePump] or Null.
Programmatic Pattern: (ObjectP[{Model[Instrument, FilterBlock], Object[Instrument, FilterBlock], Model[Instrument, PeristalticPump], Object[Instrument, PeristalticPump], Model[Instrument, VacuumPump], Object[Instrument, VacuumPump], Model[Instrument, Centrifuge], Object[Instrument, Centrifuge], Model[Instrument, SyringePump], Object[Instrument, SyringePump]}] | Automatic) | Null
Filter
The filter that should be used to remove impurities from the SamplesIn prior to starting the experiment.
Default Calculation: Will automatically resolve to a filter appropriate for the filtration type and instrument.
Pattern Description: An object of type or subtype Model[Container, Plate, Filter], Model[Container, Vessel, Filter], or Model[Item, Filter] or Null.
Programmatic Pattern: (ObjectP[{Model[Container, Plate, Filter], Model[Container, Vessel, Filter], Model[Item, Filter]}] | Automatic) | Null
FilterMaterial
The membrane material of the filter that should be used to remove impurities from the SamplesIn prior to starting the experiment.
Default Calculation: Resolves to an appropriate filter material for the given sample is Filtration is set to True.
Pattern Description: Cellulose, Cotton, Polyethylene, Polypropylene, PTFE, Nylon, PES, PLUS, PVDF, GlassFiber, GHP, UHMWPE, EPDM, DuraporePVDF, GxF, ZebaDesaltingResin, NickelResin, Silica, or HLB or Null.
PrefilterMaterial
The material from which the prefilter filtration membrane should be made of to remove impurities from the SamplesIn prior to starting the experiment.
Pattern Description: Cellulose, Cotton, Polyethylene, Polypropylene, PTFE, Nylon, PES, PLUS, PVDF, GlassFiber, GHP, UHMWPE, EPDM, DuraporePVDF, GxF, ZebaDesaltingResin, NickelResin, Silica, or HLB or Null.
FilterPoreSize
The pore size of the filter that should be used when removing impurities from the SamplesIn prior to starting the experiment.
Default Calculation: Resolves to an appropriate filter pore size for the given sample is Filtration is set to True.
Pattern Description: 0.008 micrometers, 0.1 micrometers, 0.2 micrometers, 0.22 micrometers, 0.45 micrometers, 1. micrometer, 1.1 micrometers, 2.5 micrometers, 6. micrometers, 20. micrometers, 30. micrometers, or 100. micrometers or Null.
PrefilterPoreSize
The pore size of the filter; all particles larger than this should be removed during the filtration.
Pattern Description: 0.008 micrometers, 0.1 micrometers, 0.2 micrometers, 0.22 micrometers, 0.45 micrometers, 1. micrometer, 1.1 micrometers, 2.5 micrometers, 6. micrometers, 20. micrometers, 30. micrometers, or 100. micrometers or Null.
FilterSyringe
Default Calculation: Resolves to an syringe appropriate to the volume of sample being filtered, if Filtration is set to True.
Pattern Description: An object of type or subtype Model[Container, Syringe] or Object[Container, Syringe] or a prepared sample or Null.
Programmatic Pattern: ((ObjectP[{Model[Container, Syringe], Object[Container, Syringe]}] | _String) | Automatic) | Null
FilterHousing
The filter housing that should be used to hold the filter membrane when filtration is performed using a standalone filter membrane.
Default Calculation: Resolve to an housing capable of holding the size of the membrane being used, if filter with Membrane FilterType is being used and Filtration is set to True.
Pattern Description: An object of type or subtype Model[Instrument, FilterHousing], Object[Instrument, FilterHousing], Model[Instrument, FilterBlock], or Object[Instrument, FilterBlock] or Null.
Programmatic Pattern: (ObjectP[{Model[Instrument, FilterHousing], Object[Instrument, FilterHousing], Model[Instrument, FilterBlock], Object[Instrument, FilterBlock]}] | Automatic) | Null
FilterIntensity
Default Calculation: Will automatically resolve to 2000 GravitationalAcceleration if FiltrationType is Centrifuge and Filtration is True.
Pattern Description: Greater than 0 revolutions per minute or greater than 0 standard accelerations due to gravity on the surface of the earth or Null.
Programmatic Pattern: ((GreaterP[0*RPM] | GreaterP[0*GravitationalAcceleration]) | Automatic) | Null
FilterTime
Default Calculation: Will automatically resolve to 5 Minute if FiltrationType is Centrifuge and Filtration is True.
FilterTemperature
The temperature at which the centrifuge chamber will be held while the samples are being centrifuged during filtration.
Default Calculation: Will automatically resolve to 22 Celsius if FiltrationType is Centrifuge and Filtration is True.
FilterContainerOut
The desired container filtered samples should be produced in or transferred into by the end of filtration, with indices indicating grouping of samples in the same plates, if desired.
Default Calculation: Automatically set as the PreferredContainer for the Volume of the sample. For plates, attempts to fill all wells of a single plate with the same model before using another one.
Pattern Description: An object of type or subtype Model[Container] or Object[Container] or a prepared sample or {Index, Container} or Null.
Programmatic Pattern: (((ObjectP[{Model[Container], Object[Container]}] | _String) | {GreaterEqualP[1, 1] | Automatic, (ObjectP[{Model[Container], Object[Container]}] | _String) | Automatic}) | Automatic) | Null
FilterAliquotDestinationWell
The desired position in the corresponding AliquotContainer in which the aliquot samples will be placed.
Default Calculation: Automatically resolves to A1 in containers with only one position. For plates, fills wells in the order provided by the function AllWells.
FilterAliquotContainer
The desired type of container that should be used to prepare and house the filter samples which should be used in lieu of the SamplesIn for the experiment.
Programmatic Pattern: ((ObjectP[Model[Container]] | {GreaterEqualP[1, 1] | (Automatic | Null), (ObjectP[{Model[Container], Object[Container]}] | _String) | Automatic}) | Automatic) | Null
FilterAliquot
The amount of each sample that should be transferred from the SamplesIn into the FilterAliquotContainer when performing an aliquot before filtration.
Default Calculation: Automatically set as the smaller between the current sample volume and the maximum volume of the destination container.
Pattern Description: All or greater than or equal to 1 microliter and less than or equal to 20 liters or Null.
FilterSterile
Default Calculation: Resolve to False if Filtration is indicated. If sterile filtration is desired, this option must manually be set to True.
Aliquoting
Aliquot
Indicates if aliquots should be taken from the SamplesIn and transferred into new AliquotSamples used in lieu of the SamplesIn for the experiment. Note that if NumberOfReplicates is specified this indicates that the input samples will also be aliquoted that number of times. Note that Aliquoting (if specified) occurs after any Sample Preparation (if specified).
AliquotAmount
Default Calculation: Automatically set as the smaller between the current sample volume and the maximum volume of the destination container if a liquid, or the current Mass or Count if a solid or counted item, respectively.
Programmatic Pattern: ((RangeP[1*Microliter, 20*Liter] | RangeP[1*Milligram, 20*Kilogram] | GreaterP[0*Unit, 1*Unit] | GreaterP[0., 1.] | All) | Automatic) | Null
TargetConcentration
The desired final concentration of analyte in the AliquotSamples after dilution of aliquots of SamplesIn with the ConcentratedBuffer and BufferDiluent which should be used in lieu of the SamplesIn for the experiment.
TargetConcentrationAnalyte
Default Calculation: Automatically set to the first value in the Analytes field of the input sample, or, if not populated, to the first analyte in the Composition field of the input sample, or if none exist, the first identity model of any kind in the Composition field.
Pattern Description: An object of type or subtype Model[Molecule], Model[Molecule, cDNA], Model[Molecule, Oligomer], Model[Molecule, Transcript], Model[Molecule, Protein], Model[Molecule, Protein, Antibody], Model[Molecule, Carbohydrate], Model[Molecule, Polymer], Model[Resin], Model[Resin, SolidPhaseSupport], Model[Lysate], Model[ProprietaryFormulation], Model[Virus], Model[Cell], Model[Cell, Mammalian], Model[Cell, Bacteria], Model[Cell, Yeast], Model[Tissue], Model[Material], or Model[Species] or Null.
AssayVolume
Default Calculation: Automatically determined based on Volume and TargetConcentration option values.
Pattern Description: Greater than or equal to 1 microliter and less than or equal to 20 liters or Null.
ConcentratedBuffer
The concentrated buffer which should be diluted by the BufferDilutionFactor in the final solution (i.e., the combination of the sample, ConcentratedBuffer, and BufferDiluent). The ConcentratedBuffer and BufferDiluent will be combined and then mixed with the sample, where the combined volume of these buffers is the difference between the AliquotAmount and the total AssayVolume.
Pattern Description: An object of type or subtype Model[Sample] or Object[Sample] or a prepared sample or Null.
BufferDilutionFactor
The dilution factor by which the concentrated buffer should be diluted in the final solution (i.e., the combination of the sample, ConcentratedBuffer, and BufferDiluent). The ConcentratedBuffer and BufferDiluent will be combined and then mixed with the sample, where the combined volume of these buffers is the difference between the AliquotAmount and the total AssayVolume.
Default Calculation: If ConcentratedBuffer is specified, automatically set to the ConcentratedBufferDilutionFactor of that sample; otherwise, set to Null.
BufferDiluent
The buffer used to dilute the aliquot sample such that ConcentratedBuffer is diluted by BufferDilutionFactor in the final solution. The ConcentratedBuffer and BufferDiluent will be combined and then mixed with the sample, where the combined volume of these buffers is the difference between the AliquotAmount and the total AssayVolume.
Default Calculation: Automatically resolves to Model[Sample, "Milli-Q water"] if ConcentratedBuffer is specified; otherwise, resolves to Null.
Pattern Description: An object of type or subtype Model[Sample] or Object[Sample] or a prepared sample or Null.
AssayBuffer
The buffer that should be added to any aliquots requiring dilution, where the volume of this buffer added is the difference between the AliquotAmount and the total AssayVolume.
Default Calculation: Automatically resolves to Model[Sample, "Milli-Q water"] if ConcentratedBuffer is not specified; otherwise, resolves to Null.
Pattern Description: An object of type or subtype Model[Sample] or Object[Sample] or a prepared sample or Null.
AliquotSampleStorageCondition
The non-default conditions under which any aliquot samples generated by this experiment should be stored after the protocol is completed.
Pattern Description: {AmbientStorage, Refrigerator, Freezer, DeepFreezer, CryogenicStorage, YeastIncubation, YeastShakingIncubation, BacterialIncubation, BacterialShakingIncubation, MammalianIncubation, ViralIncubation, CrystalIncubation, AcceleratedTesting, IntermediateTesting, LongTermTesting, UVVisLightTesting} or Disposal or Null.
DestinationWell
The desired position in the corresponding AliquotContainer in which the aliquot samples will be placed.
Default Calculation: Automatically resolves to A1 in containers with only one position. For plates, fills wells in the order provided by the function AllWells.
Pattern Description: Any well from A1 to H12 or list of one or more any well from A1 to H12 or any well from A1 to H12 entries or Null.
Programmatic Pattern: ((WellPositionP | {((Automatic | Null) | WellPositionP)..}) | Automatic) | Null
AliquotContainer
The desired type of container that should be used to prepare and house the aliquot samples, with indices indicating grouping of samples in the same plates, if desired. This option will resolve to be the length of the SamplesIn * NumberOfReplicates.
Default Calculation: Automatically set as the PreferredContainer for the AssayVolume of the sample. For plates, attempts to fill all wells of a single plate with the same model before aliquoting into the next.
Pattern Description: An object of type or subtype Model[Container] or Object[Container] or a prepared sample or Automatic or Null or {Index, Container} or list of one or more an object of type or subtype Model[Container] or Object[Container] or a prepared sample or Automatic or Null entries or list of one or more Automatic or Null or {Index, Container} entries.
Programmatic Pattern: (((ObjectP[{Model[Container], Object[Container]}] | _String) | (Automatic | Null) | {GreaterEqualP[1, 1] | (Automatic | Null), (ObjectP[{Model[Container], Object[Container]}] | _String) | (Automatic | Null)} | {((ObjectP[{Model[Container], Object[Container]}] | _String) | (Automatic | Null))..} | {({GreaterEqualP[1, 1] | (Automatic | Null), (ObjectP[{Model[Container], Object[Container]}] | _String) | (Automatic | Null)} | (Automatic | Null))..}) | Automatic) | Null
AliquotPreparation
Default Calculation: Automatic resolution will occur based on manipulation volumes and container types.
ConsolidateAliquots
Protocol Options
Organizational Information
Template
A template protocol whose methodology should be reproduced in running this experiment. Option values will be inherited from the template protocol, but can be individually overridden by directly specifying values for those options to this Experiment function.
Pattern Description: An object of type or subtype Object[Protocol] or an object of type or subtype of Object[Protocol] with UnresolvedOptions, ResolvedOptions specified or Null.
Programmatic Pattern: (ObjectP[Object[Protocol]] | FieldReferenceP[Object[Protocol], {UnresolvedOptions, ResolvedOptions}]) | Null
Name
A object name which should be used to refer to the output object in lieu of an automatically generated ID number.
Post Experiment
MeasureWeight
Indicates if any solid samples that are modified in the course of the experiment should have their weights measured and updated after running the experiment. Please note that public samples are weighed regardless of the value of this option.
MeasureVolume
Indicates if any liquid samples that are modified in the course of the experiment should have their volumes measured and updated after running the experiment. Please note that public samples are volume measured regardless of the value of this option.
ImageSample
Example Calls
MALDI Mass Spectrometry
Use MALDI mass spectrometry to acquire mass spectra. Provide the calibrant and the matrix to use, and indicate that during spotting of the MALDIPlate every other well is skipped in order to decrease any cross-contamination risks between samples:
Use MALDI mass spectrometry to acquire mass spectra. Provide optimized ionization parameters for your samples of particular analyte type and mass scan range:
ESI Mass Spectrometry (both TripleQuadrupole and QTOF detectors)
Use ESI and DirectInfusion mass spectrometry to acquire mass spectra. The sample is connected directly to the fluidics deck of the mass spectrometer. First, the QTOF instrument's mass calibration is performed to ensure optimal mass accuracy. Then, for the duration of RunDuration, the sample will be infused directly from the fluidics deck into the mass spectrometer leading to a constant signal over time:
Use ESI and flow injection mass spectrometry to acquire mass spectra. The sample is placed into the autosampler of the affiliated LC system. After mass calibration of the QTOF, the selected InjectionVolume is picked up from the sample by the autosampler needle, sandwiched in the chosen Buffer and injected into the mass spectrometer at InfusionFlowRate. The resulting mass spectrum will have a peak of signal when the sample reaches the detector (at around 20-30 Seconds given the flow rate provided here):
Optimize the signal intensity of your sample. Increasing the ScanTime (or DwellTime) allows more ions to reach the detector for each particular spectral acquisition thus increasing signal intensity. Ionization parameters must always be adjusted according to the sample properties in order to achieve optimal sensitivity:
Tandem Mass-Spectrometry
Toggle the dissociation of ions and control the intensity of the collision. Select fragment ions to detection:
Change the ScanMode (the alternation of selection for both fragment and intact ions). For the case of MultipleReactionMonitoring, specific intact and fragment ions can be specified for measurement:
Change the ScanMode (the alternation of selection for both fragment and intact ions). For the case of NeutralIonLoss, the NeutralLoss field can be used to track the loss e loss of small molecules (such as water, ammonia, or carbon monoxide) during intact ion fragmentation.
Preferred Input Containers
ESI-QTOF Direct Infusion: the fluidics deck of the QTOF supports 2mL tubes (1mL - 1.5mL volume) and 30mL reservoirs (2mL to 30mL volume). The sample amount needed is the sum of dead volume (1mL and 2mL respectively) and RunDuration * InfusionFlowRate.
ESI-QQQ Direct Infusion: any container that the solution inside can be drawn by a syringe. The sample amount needed is InfusionVolume. For a very large-volume sample, transferring to a small-volume tube (e.g., 2mL skirted tube) is recommended.
ESI Flow Injection (for Both ESI-QQQ and ESI QTOF): for flow injection, samples are loaded into the LC autosampler which supports 2mL HPLC vials and regular 96 deep well plates. The sample amount used is the sum of dead volume (20 ul) and RunDuration * InfusionFlowRate.
MALDI-TOF: Any container that fit on the robotic liquid handler decks, where the spotting of the target plate takes place, is supported. For small-volume samples, the use of skirted tubes or plates with V-shaped bottom is recommended (see a non-complete list of examples below).
Data Processing
Plot the mass spectra generated from a ESI mass spectrometry experiment of a single sample, with pink ticks as the expected molecular weight:
Use the ExpectedMolecularWeight, TickColor, TickStyle and TickSize options to set the position and style of ticks for the molecular weight that is supposed to show up in this figure:
Warnings and Errors
Messages (80)
AliquotOptionConflict (1)
AliquotRequired (1)
AutoNeutralLossValueWarnings (1)
AutoResolvedFragmentMassDetectionFixedValues (1)
AutoResolvedMassDetectionFixedValue (1)
CalibrantIncompatibleWithIonSource (2)
CalibrantMassDetectionMismatch (1)
DefaultMassDetection (1)
DirectInfusionUnneededOptions (2)
ESITripleQuadTooManyCalibrants (1)
ExceedsMALDIPlateCapacity (1)
In MALDI mass spectrometry measurements, if the total number of spots required, given calibrant spots and matrix control spots, exceeds the number of wells on the MALDI plate, an experiment cannot be generated. Note that the number of matrix and calibrant spots can be decreased by using fewer unique settings:
FilteredAnalytes (1)
FlowInjectionRequiredOptions (2)
FlowInjectionUnneededOptions (1)
FragmentMassDetectionScanModeMismatches (1)
IncompatibleCalibrant (1)
IncompatibleInstrument (1)
IncompatibleMassAnalyzerAndInstrument (1)
IncompatibleMassAnalyzerAndIonSource (1)
IncompatibleMassDetection (1)
InfusionVolumeLessThanRunDurationTimesFlowRate (1)
InputOptionsMRMAssaysMismatches (1)
InstrumentPrecision (3)
The precision of the ESICapillaryVoltage cannot be not more than 0.01 Kilovolt, the maximum precision achievable by the instrumentation:
The precision of the SampleVolume cannot be not more than 0.1 Microliter, the maximum precision achievable by ECL liquid handlers:
The precision of the SpottingDryTime cannot be not more than 1 minute, the maximum precision achievable by ECL liquid handlers :
InvalidCalibrantLaserPowerRange (1)
InvalidCalibrantMatrixSample (1)
InvalidDesolvationGasFlows (1)
InvalidESIQTOFGasOption (1)
InvalidESIQTOFMassDetectionOption (1)
InvalidESIQTOFVoltagesOption (1)
InvalidLaserPowerRange (1)
invalidMALDITOFMassDetectionOption (1)
InvalidMassToleranceInputs (1)
InvalidMatrixSample (1)
InvalidMultipleReactionMonitoringLengthOfInputOptions (1)
LimitedReferencePeaks (1)
LowInMass (1)
LowMinMass (1)
MALDICalibrantNumberOfShotsTooSmall (1)
MALDINumberOfShotsTooSmall (1)
MassDetectionScanModeMismatches (1)
MassSpecRequiredOptions (2)
MassSpectrometryIncompatibleAliquotContainer (1)
MassSpectrometryInvalidCalibrants (3)
For MALDI-TOF,Calibrants that are deprecated and without ReferencePeaksPositiveMode and ReferencePeaksNegativeMode filled cannot be used:
For ESI-QTOF, Calibrants that are deprecated and without ReferencePeaksPositiveMode and ReferencePeaksNegativeMode filled cannot be used:
For ESI-QQQ, Calibrants that are deprecated and without ReferencePeaksPositiveMode and ReferencePeaksNegativeMode filled cannot be used:
OutRangedDesolvationTemperature (2)
SamplesOutOfMassDetection (1)
SpottingInstrumentIncompatibleAliquots (1)
TooManyESISamples (1)
TooManyMALDISamples (1)
TooManyMultpleReactionMonitoringAssays (1)
TooShortRunDurations (1)
UnableToCalibrate (1)
UninformedCalibrantMatrix (1)
UninformedMatrix (1)
UnknownMolecularWeight (1)
UnneededOptions (1)
UnneededTandemMassSpecOptions (2)
UnsupportedMALDIPlate (1)
Possible Issues
Choice of MALDI matrix
In MALDI experiments, the choice of right matrix is key to successful ionization. In general, highly polar analytes are preferably combined with highly polar matrices, and non-polar analytes work better with non-polar matrices.
Signal optimization in MALDI experiments
In MALDI mass spectrometry experiments, different matrices and sample types (in particular different mass scan ranges) require different laser and voltage settings to achieve optimal signal intensity, resolution and signal-to-noise ratio. You may use the following rough optimization strategy: If the signal-to-noise ratio is low, adjust the acceleration voltage and the number of shots. If the signal intensity is low, increase the laser power. Note that excessive laser power may result in saturated peaks with poor resolution and high sample consumption. If the mass resolution is low (peaks are wide), adjust the delay time and the grid voltage. In general, analytes with higher m/z require longer delay times and lower grid voltages, while analytes with smaller m/z need shorter delay time and higher grid voltage for optimal performance.
Signal optimization in ESI experiments
In ESI mass spectrometry, different sample types and flow rates require different source settings. You may follow these guidelines for signal optimization: If the signal intensity is too high, lower the capillary voltage and the injection flow rate, or dilute the samples. If the sensitivity is too low, adjust the capillary and sample cone voltage and temperature, or increase injected sample amount. Note that sometimes matrix effects can occur, so in rare cases, a dilution of the sample can paradoxically lead to an increase in sensitivity. If the signal-to-noise ratio is low, lower the scan rate and increase the injection flow rate, and inspect your buffer for contaminants that may cause an increased baseline.
Last modified on Mon 12 Aug 2024 22:26:46