Experimental conditions following MIAPE guidelines


Experimental conditionsActivationSample PreparationQuantificationMSMS InformaticsExperiment IDs
Qualitative analysis (Resting T-cells). SDS-PAGE. RestingS01-MS1MSI120, 21
Qualitative analysis (Resting T-cells). Classical digestion. SCX. RestingS02-MS2MSI119, 24
Controls (Resting T-cells). iTRAQ labeling. RestingS03Q1MS1MSI23
Activation PMA-ionomicine, 4h. iTRAQ labeling. 4-h PMA/IonomycinS04Q1MS1MSI21, 2
Controls (Resting T-cells). TMT labeling. Classical digestion. RestingS05Q2MS2MSI34
Activation PMA-ionomicine, 15min and 2h. TMT labeling. Classical digestion. 15-min and 2-h PMA/IonomycinS06Q2MS2MSI36, 7
Qualitative analysis (Resting T-cells). FASP digestion. SCX. RestingS07-MS2MSI127, 31, 32, 33, 34
Qualitative analysis (Resting T-cells). Classical digestion. OGE. RestingS08-MS2MSI18, 9, 10, 11, 12, 13, 14
Qualitative analysis (Resting T-cells). FASP digestion. OGE. RestingS09-MS2MSI115, 16, 17, 18
Controls (Resting T-cells). TMT labeling. FASP digestion. RestingS10Q2MS2MSI328
Activation PMA-ionomicine, 15min and 2h. TMT labeling. FASP digestion. 15-min and 2-h PMA/IonomycinS11Q2MS2MSI329, 30, 36
T-primary cells. Activation with antiCD3/CD28. 15min and 2h. TMT labeling. 15-min and 2-h antiCD3/CD28S12Q2MS2MSI335


S01
Sample Preparation for Phosphoproteomics 2008/8/08
1.General features
1.1.Date stamp24/07/2008
1.2.Responsible person or institutional roleDra. Montserrat Carrascal. CSIC/UAB Proteomics Facility, IIBB-CSIC, Building M, Campus UAB, 08193 Bellaterra, Spain
1.3.Sample nameResting T-lymphocytes
2.Sample processing
2.1.Culture/tissue handling
2.1.1.Tissue/Cell/Fluid typeT-lymphocytes
SourceBuffy coat
SpeciesHuman
Cell Isolation MethodFicoll-Paque gradient centrifugation
Cell/Pellet lysis-
Buffer6 M urea, 50 mM Tris oH 8.5, phosphatase inhibitors, 2 mM DTT, 2 x 108 cell / mL
Homogenization or sonication methodsSonication (Sonic Vibracell TM), 5s x 5 times, 60% power
Centrifugation30 min / 2000xg
Protein Precipitation10% TCA added 1/1 v/v to sample, 1 h incubation, wash with 1 mL acetone
2.2.Digestion
2.2.1Pretreatment
Reduction2.5 mM DTT, 30 min, 56oC
Alkykation7 mM IAA, 30 min, 20oC
OthersOther treatments before digestion. Include reagents, time, temperature and concentration.
2.2.2.Digestion parameters
3.Protein Purification/Fractionation
3.1.SDS-PAGE and in-gel digestion (from HUPO-PSI MIAPE:Gel Electrophoresis vs 1.4, January 2008)
3.1.1Gel matrix and electrophoresis
Description of gel matrixSDS-PAGE
Gel manufacturerIn-house prepared gel. (Laemmli, U.K.. Nature 227, 680-685, (1970))
Physical dimensionsMini-Protean (BioRad)
Physicochemical property range and distribution-
Acrylamide concentrationStaking gel 4%, running gel 9%
Acrylamide:crosslinker ratioThe acrylamide to bisacrylamide ratio of the gel (Name of crosslinker and ratio described as "acrylamide:crosslinker")
Gel lane1
Sample applicationName of sample: Resting_Lymphocytes, Volume of loading buffer, 900 uL
Buffer25 mM Tris-base, 192 mM Glycine, 0.1% SDS
EquipmentMiniProtean-II, BioRad
Electrophoresis conditions2 h / 30 V, 1h / 100 V
3.1.2Staining
DescriptionCoomassie Blue
3.1.3In gel digestion
EnzymeTrypsin (Promega), 20 ng/uL, 50 uL
Incubation parametersIncubation time and temperature
Previous treatmentReduction and alkylation
Automatic digestorIntavis MS Pro
5.Phosphopeptide Enrichment
5.1.Method 1
Method descriptionIMAC
Loading buffer250 mM Acetic Acid / 30% ACN
ResinPhos-Select (Sigma)
Incubation time90 min / 20oC
Washing buffer250 mM Acetic Acid / 30% ACN
Elution buffer0.5% NH4OH (neutralized with formic acid)
Other parametersEvaporated to dryness and redisolved in 40 uL 0.1% FA for LC-MS
5.2.Method 2
Method descriptionTiO2
Loading buffer1 M glycolic acid, 5% TFA, 80% ACN
ResinTitansphere, GL Sciences, Japan
Incubation time-
Washing buffer1 M glycolic acid, 5% TFA, 80% ACN, 80% ACN 1% TFA
Elution buffer0.5% NH4OH
Other parametersPerform in a hand-made tip column. Evaporated to dryness and redisolved in 40 uL 0.1% FA for LC/MS

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S02  
Sample Preparation for Phosphoproteomics 2008/8/08
1General features
1.1.Date stamp04/08/2008
1.2.Responsible person or institutional roleDra. Montserrat Carrascal. CSIC/UAB Proteomics Facility, IIBB-CSIC, Building M, Campus UAB, 08193 Bellaterra, Spain
1.3.Sample nameResting T-lymphocytes
2.Sample processing
2.1.Culture/tissue handling
2.1.1.Tissue/Cell/Fluid typeT-lymphocytes
SourceBuffy coat
SpeciesHuman
Cell Isolation MethodFicoll-Paque gradient centrifugation
Cell/Pellet lysis-
Buffer6 M urea, 50 mM Tris oH 8.5, phosphatase inhibitors, 2 mM DTT, 2 x 108 cell / mL
Homogenization or sonication methodsSonication (Sonic Vibracell TM), 5s x 5 times, 60% power
Centrifugation30 min / 2000xg
Protein Precipitation10% TCA added 1/1 v/v to sample, 1 h incubation, wash with 1 mL acetone
2.2.Digestion
2.2.1Pretreatment
Reduction2.5 mM DTT, 30 min, 56oC
Alkykation7 mM IAA, 30 min, 20oC
Others-
2.2.2Digestion parameters
Protein concentration1 mg/mL
Buffer1.8 M urea, 15 mM Tris oH 8.5, phosphatase inhibitors, 2mM DTT, 25 mM ammonium bicarbonate
EnzymeTrypsin (Sigma)
Incubation parameters24 h
2.4.Other treatments
DescriptionSample desalting with tC18 cartriges
4.Peptide Purification/Fractionation
4.1.HPLC (from MIAPE:Column Chromatography vs 1.0, June 2008)
4.1.1Equipment
4.1.1.1Product details for column
MakeHewlett Packard
ModelAgilent 1100
Separation modeStrong Cation Exchange
4.1.1.2Physical characteristics of column
Length200mm
Diameter2.1mm
Description of stationary phasePolysulfoethyl A,200 Angstrom
4.1.1.3Equipment: Chromatography system used for separation
Manufacturer and modelAgilent 1100 (binary pump, UV detector and manual injector)
4.1.2Mobile phase
Names of mobile phaseA and B
Description of the constituentsA: mQ 0.1% formic (70%) ACN (30%),B: mQ 0.1% formic (70%) 500mM NH4Cl, ACN (30%)
4.1.3Properties of the column run
Time80 min
Gradient0 min 0%B, 3 min 0%B,38 min 25%B,58min 100%B,78 min 100%B,80min 0%B
Flow rate200uL/min
Temperature20o C
Separation purposePeptide Fractionation
4.1.4.1Column outputs: Detection
Equipment used for detectionUV Lamp
Typemicro cell
Equipment settings220 nm
Timescale over which data was collected0 min to 80 min
4.1.4.2Column outputs: Fractions
Fraction nameSCX1_15 to SCX1_63
Fraction descriptionFractions were collected each 3 minutes from minute 15
5.Phosphopeptide Enrichment
5.1.Method 1
Method descriptionIMAC
Loading buffer250 mM Acetic Acid / 30% ACN
ResinPhos-Select (Sigma)
Incubation time90 min / 20oC
Washing buffer250 mM Acetic Acid / 30% ACN
Elution buffer0.5% NH4OH (neutralized with formic acid)
Other parametersEvaporated to dryness and redisolved in 40 uL 0.1% FA for LC-MS
5.2.Method 2
Method descriptionTiO2
Loading buffer1 M glycolic acid, 5% TFA, 80% ACN
ResinTitansphere, GL Sciences, Japan
Incubation time-
Washing buffer1 M glycolic acid, 5% TFA, 80% ACN, 80% ACN 1% TFA
Elution buffer0.5% NH4OH
Other parametersPerform in a hand-made tip column. Evaporated to dryness and redisolved in 40 uL 0.1% FA for LC/MS

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S03
 Sample Preparation for Phosphoproteomics 2008/8/08
1General features
1.1.Date stamp04/08/2008
1.2.Responsible person or institutional roleDra. Montserrat Carrascal. CSIC/UAB Proteomics Facility, IIBB-CSIC, Building M, Campus UAB, 08193 Bellaterra, Spain
1.3.Sample nameResting T-lymphocytes
2.Sample processing
2.1.Culture/tissue handling
2.1.1.Tissue/Cell/Fluid typeT-lymphocytes
SourceBuffy coat
SpeciesHuman
Cell Isolation MethodFicoll-Paque gradient centrifugation
Cell/Pellet lysis-
Buffer6 M urea, 50 mM Tris oH 8.5, phosphatase inhibitors, 2 mM DTT, 2 x 108 cell / mL
Homogenization or sonication methodsSonication (Sonic Vibracell TM), 5s x 5 times, 60% power
Centrifugation30 min / 2000xg
Protein Precipitation10% TCA added 1/1 v/v to sample, 1 h incubation, wash with 1 mL acetone
2.2.Digestion
2.2.1Pretreatment
Reduction2.5 mM DTT, 30 min, 56oC
Alkykation7 mM IAA, 30 min, 20oC
Others-
2.2.2Digestion parameters
Protein concentration1 mg/mL
Buffer1.8 M urea, 15 mM Tris oH 8.5, phosphatase inhibitors, 2mM DTT, 25 mM ammonium bicarbonate
EnzymeTrypsin (Sigma)
Incubation parameters24 h
2.4.Other treatments
DescriptionSample desalting with tC18 cartriges
4.Peptide Purification/Fractionation
4.1.HPLC (from MIAPE:Column Chromatography vs 1.0, June 2008)
4.1.1Equipment
4.1.1.1Product details for column
MakeHewlett Packard
ModelAgilent 1100
Separation modeStrong Cation Exchange
4.1.1.2Physical characteristics of column
Length200mm
Diameter2.1mm
Description of stationary phasePolysulfoethyl A,200 Angstrom
4.1.1.3Equipment: Chromatography system used for separation
Manufacturer and modelAgilent 1100 (binary pump, UV detector and manual injector)
4.1.2Mobile phase
Names of mobile phaseA and B
Description of the constituentsA: mQ 0.1% formic (70%) ACN (30%),B: mQ 0.1% formic (70%) 500mM NH4Cl, ACN (30%)
4.1.3Properties of the column run
Time80 min
Gradient0 min 0%B, 3 min 0%B,38 min 25%B,58min 100%B,78 min 100%B,80min 0%B
Flow rate200uL/min
Temperature20o C
Separation purposePeptide Fractionation
4.1.4.1Column outputs: Detection
Equipment used for detectionUV Lamp
Typemicro cell
Equipment settings220 nm
Timescale over which data was collected0 min to 80 min
4.1.4.2Column outputs: Fractions
Fraction nameSCX1_15 to SCX1_63
Fraction descriptionFractions were collected each 3 minutes from minute 15
5.Phosphopeptide Enrichment
5.1.Method 1
Method descriptionTiO2
Loading buffer1 M glycolic acid, 5% TFA, 80% ACN
ResinTitansphere, GL Sciences, Japan
Incubation time-
Washing buffer1 M glycolic acid, 5% TFA, 80% ACN, 80% ACN 1% TFA
Elution buffer0.5% NH4OH
Other parametersPerform in a hand-made tip column. Evaporated to dryness and redisolved in 40 uL 0.1% FA for LC/MS

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 MS1 
 HUPO-PSI MIAPE: Mass Spectrometry MS v2.24
1.General features
1.a.Global descriptors
1.a.1Machine manufacturer, modelLineal Ion Trap (LTQ, Thermo Fisher)
1.a.2Estimated mass accuracy (ppm or Dalton) for all MS levels for which data are presented0.3-0.6 Da
1.b.Control and analysis software
1.b.1Software name and versionXcalibur 2.0
1.b.2Switching criteria:Dependent data analysis with neutral loss (MS and 8 MS/MS)
1.b.3Isolation width3 units (m/z)
2.Ion sources
2.a.Electrospray Ionisation (ESI)
2.a.1Supply typeChromatography
2.a.2Scan cycle times50 ms all levels (average)
2.a.3Solvent flow rate and composition:solvent A: Water 0.1% formic acid, solvent B: Acetonitrile 0.1% Formic Acid
2.a.4Interface manufacturer, modelmicroESI from Thermo Fisher
2.a.5Sprayer type, coating, manufacturer, modelSilica Tips, New Objective, FS360-100-30-CE-5
2.a.6Relevant voltages where appropriate (tip,cone,acceleration)Tubel lens = 130 V, cap lens = 48 V, spray voltage = 2 kV
2.a.7Whether in-source dissociation performed:No
3.Post-source componentry
3.c.Ion trap
3.c.1Final MS exponent achievedMS^3
3.d.Collision cell
3.d.1Gas type and pressure (bar)unknow
3.d.2Collision energy0.35
3.f.Detectors 
3.f.1Detector typeunknow
3.f.2Detector sensitivityunknow
4.Peak list generation and annotation (a) Generation of spectrum
4.a.1Location of source file including file name and typeThermo RAW binary file generated by Xcalibur
4.a.2Parameters triggering the generation of peak lists from raw dataPeak list generated in 600-4500 amu range. Precursor mass tolerance for grouping: 1.4. Intensity threshold set to minimum (1 absolute intensity). Minimum ion count for peaklist generation: 10. Related grouped scans needed for a .dta file generation: 1.
4.a.3Signal-to-noise estimation and methodICIS algorithm: area noise factor 5, peak noise factor 10.INCOS noise method (min peak width: 3, multiplet resolution: 10, area tail extension: 5, area scan window 0)
4.a.4Smoothing; whether applied, parametersICIS algorithm: 15 smoothing points
4.a.5Percentage peak height for centroiding; or algorithm used, if appropriateICIS algorithm
4.a.6Background threshold, or algorithm usedICIS algorithm: baseline window 40
4.a.7Charge state calculationAutomatic charge state calculation: direct +1 direct assignement, +2/+3 alternatives redundantly generated
4.a.8Ion mode for this spectrumPositive

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MIAPE (Minimum Information About a Proteomics Experiment) data term definitions

These forms try to compile the minimum information required to report a phosphoproteomics experiment, in the line of the criteria applied in HUPO-PSI MIAPE guidelines.

Mass Spectrometry (MS) and Mass Spectrometry Informatics (MSI) data follow previously published MIAPE guidelines.

A sample preparation form has been implemented 'in house' for our experiments. The description is shown in the table below:


Sample preparation for Phosphoproteomics
Form version 0.90: Sample Preparation for phosphoproteomics/ 2008-08-08
1General features
1.1Date stamp (as yyyy-mm-dd)The date on which the work described was initiated; given in the standard yyyy-mm-dd format (with hyphens)
1.2Responsible person or institutional roleThe (stable) primary contact person for this data set: this could be the experimenter, lab head, line manager, etc. Where responsibility rest with an institutional role (e.g. One of a number of duty officers) rather than a person, give the official mane of the role rather than any one person. In all cases give affiliation and stable contact information, which consists of (i) Name, (ii) Postal address and (iii) Email address.
1.3Sample/s name/sName of the samples processed. Include if there are control, standard of test samples.
2. Sample processing
2.1. Culture/tissue handling
2.1.1Tissue/Cell/Fluid typeBiological material used in the experiment.
SourceSource from which the material is obtained (p.e. T-cells from buffy coat)
SpeciesSpecies of procedence
2.1.2Tissue IsolationSurgical procedure, laser capture, etc
2.1.2Cell Isolation MethodMethod used for cell isolation (e.g. Ficoll-Paque T-cells purification from buffy coat, enzymatic digestion, explant culture...)
2.1.3Culture parameters
Culture mediumGrowth medium with all the complements added
Culture conditionsTemperature and gas mixture (typically for mammalian cells, 37oC, 5% CO2)
Cell densityNumber of cells per mL
Type of cultureIndicate if there are suspension or adherent cells and if they are primary cultures
Time of cultureTime which the cells are in culture and number of previous passages
2.1.4Cell/tissue activation
Cell TreatmentIndicate cell treatment (activator,drug), including concentration and buffers used for addition
Cell densityNumber of cells per mL during treatment
Time Time for which the cells/tissues are treated.
2.1.5Pellet preparationProcedure used to prepare the pellet (wash, centrifugation or other parameters)
2.1.6Cell/Pellet lysis
BufferBuffer used for cell lysis. Indicate all the components, concentration and volume used for lysis
Homogenization or sonication methodsMethod used for cell/tissue disruption, include the time and power of the homogenization or sonication process.
CentrifugationCentrifugation time and centrifugal force
Protein PrecipitationThe method of precipitation if required. Include reagents, volumes and time of incubation
2.2.Digestion
2.2.1Pretreatment
ReductionInclude reagents, time, temperature and concentration.
AlkykationInclude reagents, time, temperature and concentration.
OthersOther treatments before digestion. Include reagents, time, temperature and concentration.
2.2.2Digestion parameters
Protein concentrationConcentration of protein extract
BufferBuffer used for digestion, indicating components, concentration and pH
EnzymeEnzyme description and concentration
Incubation parametersIncubation time and temperature
2.3. Peptide/Protein Labeling
Type of labelingType and make of the labeling reactive (e.g. iTRAQ from Applied Biosystems)
Significative customizationsAny significant deviations from the manufacture's specificated protocol
2.4. Other treatments (Other procedures or treatments performed in the sample preparation not included in 2.1.1 to 2.1.9)
3.Protein Purification/Fractionation
3.1.SDS-PAGE and in-gel digestion (from HUPO-PSI MIAPE:Gel Electrophoresis vs 1.4, January 2008)
3.1.1Gel matrix and electrophoresis
Description of gel matrixGel matrix being used. Include the descriptive name of the matrix (e.g. IPG strip, slab gel) and the type of the matrix used (e.g. a native gel, denaturing gel, gradient gel, etc.). State whether the matrix is composed of more than one kind of gel and name the parts (e.g. stacking gel). Give the dimensions of the matrix and associated parts, under the physical dimension section, below.
Gel manufacturerIf the gel was purchased pre-cast, then include the model name, model number, batch number and manufacturer. If the gel has been manufactured in house then a reference to published protocol should be given. If no published protocol is available a recipe should be given.
Physical dimensionsThe physical dimensions of the gel matrix and of any sub-matrices described in section 3.2.1. The measurements must be in the form of the Cartesian Coordinate system (x,y,z). According to the standard image orientation described in section 7.1.6, x represents the distance from the anode (+) to the cathode (-). (For example in an IPG strip x = the strip length, for a standard slab gel, x = the width). z = the matrix depth.
Physicochemical property range and distributionAs applicable, the details of the pH distribution of the matrix, including the overall pH range of the gel, if known. Details of the molecular weight distribution of matrix with appropriate measurement unit. Examples include linear pH 4-7, logarithmic apparent molecular mass 200-10 kDa.
Acrylamide concentrationThe acrylamide concentration of the gel, or each matrix as described in section 3.2.1. In the form of a single percentage (1%) or gradient (1-2%). For gradients include the gradient distribution if appropriate, (e.g. fixed, stepped or liner).
Acrylamide:crosslinker ratioThe acrylamide to bisacrylamide ratio of the gel (Name of crosslinker and ratio described as "acrylamide:crosslinker")
Gel laneThe number of lanes on the gel matrix
sample applicationDescription of the sample as applied to the matrix, giving: 1. Name of sample, Volume of loading buffer, Sample loaded per lane (if applicable for quantification, in SI measurement), and lane designation
BufferDescription of the running buffers used, in terms of name of buffer, components with concentrations.
EquipmentApparatus used for electrophoresis. Include brand and model.
Electrophoresis conditionsThe running conditions applied to the gel (To be given in terms of voltages versus time/kilovolt hours, (or appropriate measurements) and temperature).
3.1.2StainingStaining method used to develop the gel image.
3.1.3In gel digestion
EnzymeEnzyme description and concentration
Incubation parametersIncubation time and temperature
Previous treatmentPrevious treatment if required (e.g. Reduction and/or alkylation)
Automatic digestorIf required, brand and model of the apparatus used
3.2.HPLC and liquid digestion (from HUPO-PSI MIAPE:Column Chromatography vs 1.0, June 2008)
3.2.1Equipment
3.2.1.1Product details for column
MakeThe name of the manufacturer
ModelThe model number provided by the manufacturer
Separation modeA description of the type of column being used (e.g., Separation mechanism: affinity, anion exchange, cation exchange, reverse phase, size).
3.2.1.2Physical characteristics of column
LengthThe length of the column.
DiameterThe internal diameter of the column.
Description of stationary phaseA description of the constituents of the stationary phase, including the name of the packing material and the particle size.
3.2.1.3Chromatography system used for separation, where applicableThe name of the manufacturer and the model name provided by the manufacturer.
3.2.1.4Mobile phase: for each mobile phase
Name of mobile phaseName used to refer to mobile phase in Properties of Column run
Description of the constituentsFor each constituent, a description and the concentration
3.2.1.5Properties of the column run
TimeThe length of time for which the column is run. The value can be provided as a duration of time.
GradientThe proportion of each of the mobile phases at a point in time, or the function describing the gradient, including the overall duration of the gradient. There may be several steps that together make up the gradient.
Flow rateThe rate at which the mobile phase is applied to the column, including the time period for which this holds if it varies during the experiment.
TemperatureThe temperature at which the column is run, including the period for which this holds if it varies during the experiment
Separation purposeAnalytical or preparative
3.2.1.6Column outputs - (a) detection
Equipment used for detectionMake, model and description
TypeA description of the kind of detector (e.g. UV) and a description of control properties of the detector, such as the wavelength that is being detected
Equipment settingsA description of control properties of the detector, such as the wavelength that is being detected
Timescale over which data was collectedThe time range covered by the trace produced by the detector
TraceThe location and format of the trace
3.2.1.7Column outputs - (b) fractions (if required)
Fraction nameAn optional name, unique within a run, by which a fraction can be referenced.
Fraction descriptionEither a description of the procedure by which the fractions were collected (i.e. start/end time, size (time or volume), mode (fixed or peak directed), or a description of the individual fractions (e.g. time of collection, volume)
3.2.2Liquid digestion of fractions, if requiredIndicate the enzyme used, time of digestion and concentration of sample and enzyme.
EnzymeEnzyme description and concentration
Incubation parametersIncubation time and temperature
Previous treatmentPrevious treatment if requires (e.g. Reduction and/or alkylation)
3.2.3Other parametersOther parameters performed not included in 3.2.1 to 3.2.2
4.Peptide Purification/Fractionation
4.1. HPLC (from HUPO-PSI MIAPE:Column Chromatography vs 1.0, June 2008)
4.1.1Equipment
4.1.1.1Product details for column
MakeThe name of the manufacturer
ModelThe model number provided by the manufacturer
Separation modeA description of the type of column being used (e.g., Separation mechanism: affinity, anion exchange, cation exchange, reverse phase, size).
4.1.1.2Physical characteristics of column
LengthThe length of the column.
DiameterThe internal diameter of the column.
Description of stationary phaseA description of the constituents of the stationary phase, including the name of the packing material and the particle size.
4.1.1.3Chromatography system used for separationThe name of the manufacturer and the model name provided by the manufacturer.
4.1.2Mobile phase: for each mobile phase
Name of mobile phaseName used to refer to mobile phase in Properties of Column run
Description of the constituentsFor each constituent, a description and the concentration
4.1.3Properties of the column run
TimeThe length of time for which the column is run. The value can be provided as a duration of time.
GradientThe proportion of each of the mobile phases at a point in time, or the function describing the gradient, including the overall duration of the gradient. There may be several steps that together make up the gradient.
Flow rateThe rate at which the mobile phase is applied to the column, including the time period for which this holds if it varies during the experiment.
TemperatureThe temperature at which the column is run, including the period for which this holds if it varies during the experiment
Separation purposeAnalytical or preparative
4.1.4.1Column outputs - (a) detection
Equipment used for detectionMake, model and description
TypeA description of the kind of detector (e.g. UV) and a description of control properties of the detector, such as the wavelength that is being detected
Equipment settingsA description of control properties of the detector, such as the wavelength that is being detected
Timescale over which data was collectedThe time range covered by the trace produced by the detector
TraceThe location and format of the trace
4.1.4.2Column outputs - (b) fractions (if required)
Fraction nameAn optional name, unique within a run, by which a fraction can be referenced.
Fraction descriptionEither a description of the procedure by which the fractions were collected (i.e. start/end time, size (time or volume), mode (fixed or peak directed), or a description of the individual fractions (e.g. time of collection, volume)
4.1.2Other parametersOther parameters not included in 4.1.1.
5.Phosphopeptide Enrichment
5.1. Method 1
5.1.1Method descriptionBrief description of the method (e.g. IMAC purification)
5.1.2Loading bufferBuffer (type and volume) used to dissolve the sample and to load it into the resin
5.1.3ResinMaterial used for phosphopeptide enrichment. Include name of the manufacturer and reference.
5.1.4Incubation timeIncubation time of the sample with the resin
5.1.5Washing bufferBuffer (type and volume) used to wash the resin after incubation.
5.1.6Elution bufferBuffer (type and volume) used to elute the phosphopeptides from the resin.
5.1.7Other parametersOther parameters not included.

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