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Experimental conditions in MIAPE format

Experimental conditions	Activation	Sample Preparation	Fraction	MSMS	MS informatics
SDS-PAGE IMAC Resting Lymphocytes	Resting	S1	IMAC	M1	I1
SDS-PAGE TiO2 Resting Lymphocytes	Resting	S1	TiO2	M1	I1
SCX-1 IMAC with desalting Resting Lymphocytes	Resting	S2	IMAC	M1	I1
SCX-1 TiO2 with desalting Resting Lymphocytes	Resting	S2	TiO2	M1	I1
SCX-2 TiO2 Resting Lymphocytes	Resting	S3	TiO2	M1	I1

S1
Sample Preparation for Phosphoproteomics 2008/8/08
1.	General features
1.1.	Date stamp	24/07/2008
1.2.	Responsible person or institutional role	Dra. Montserrat Carrascal. CSIC/UAB Proteomics Facility, IIBB-CSIC, Building M, Campus UAB, 08193 Bellaterra, Spain
1.3.	Sample name	Resting T-lymphocytes
2.	Sample processing
2.1.	Culture/tissue handling
2.1.1.	Tissue/Cell/Fluid type	T-lymphocytes
	Source	Buffy coat
	Species	Human
	Cell Isolation Method	Ficoll-Paque gradient centrifugation
	Cell/Pellet lysis	-
	Buffer	6 M urea, 50 mM Tris oH 8.5, phosphatase inhibitors, 2 mM DTT, 2 x 108 cell / mL
	Homogenization or sonication methods	Sonication (Sonic Vibracell TM), 5s x 5 times, 60% power
	Centrifugation	30 min / 2000xg
	Protein Precipitation	10% TCA added 1/1 v/v to sample, 1 h incubation, wash with 1 mL acetone
2.2.	Digestion
2.2.1	Pretreatment
	Reduction	2.5 mM DTT, 30 min, 56oC
	Alkykation	7 mM IAA, 30 min, 20oC
	Others	Other 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)
3.1.1	Gel matrix and electrophoresis
	Description of gel matrix	SDS-PAGE
	Gel manufacturer	In-house prepared gel. (Laemmli, U.K.. Nature 227, 680-685, (1970))
	Physical dimensions	Mini-Protean (BioRad)
	Physicochemical property range and distribution	-
	Acrylamide concentration	Staking gel 4%, running gel 9%
	Acrylamide:crosslinker ratio	The acrylamide to bisacrylamide ratio of the gel (Name of crosslinker and ratio described as "acrylamide:crosslinker")
	Gel lane	1
	Sample application	Name of sample: Resting_Lymphocytes, Volume of loading buffer, 900 uL
	Buffer	25 mM Tris-base, 192 mM Glycine, 0.1% SDS
	Equipment	MiniProtean-II, BioRad
	Electrophoresis conditions	2 h / 30 V, 1h / 100 V
3.1.2	Staining
	Description	Coomassie Blue
3.1.3	In gel digestion
	Enzyme	Trypsin (Promega), 20 ng/uL, 50 uL
	Incubation parameters	Incubation time and temperature
	Previous treatment	Reduction and alkylation
	Automatic digestor	Intavis MS Pro
5.	Phosphopeptide Enrichment
5.1.	Method 1
	Method description	IMAC
	Loading buffer	250 mM Acetic Acid / 30% ACN
	Resin	Phos-Select (Sigma)
	Incubation time	90 min / 20oC
	Washing buffer	250 mM Acetic Acid / 30% ACN
	Elution buffer	0.5% NH4OH (neutralized with formic acid)
	Other parameters	Evaporated to dryness and redisolved in 40 uL 0.1% FA for LC-MS
5.2.	Method 2
	Method description	TiO2
	Loading buffer	1 M glycolic acid, 5% TFA, 80% ACN
	Resin	Titansphere, GL Sciences, Japan
	Incubation time	-
	Washing buffer	1 M glycolic acid, 5% TFA, 80% ACN, 80% ACN 1% TFA
	Elution buffer	0.5% NH4OH
	Other parameters	Perform in a hand-made tip column. Evaporated to dryness and redisolved in 40 uL 0.1% FA for LC/MS

S2
Sample Preparation for Phosphoproteomics 2008/8/08
1	General features
1.1.	Date stamp	04/08/2008
1.2.	Responsible person or institutional role	Dra. Montserrat Carrascal. CSIC/UAB Proteomics Facility, IIBB-CSIC, Building M, Campus UAB, 08193 Bellaterra, Spain
1.3.	Sample name	Resting T-lymphocytes
2.	Sample processing
2.1.	Culture/tissue handling
2.1.1.	Tissue/Cell/Fluid type	T-lymphocytes
	Source	Buffy coat
	Species	Human
	Cell Isolation Method	Ficoll-Paque gradient centrifugation
	Cell/Pellet lysis	-
	Buffer	6 M urea, 50 mM Tris oH 8.5, phosphatase inhibitors, 2 mM DTT, 2 x 108 cell / mL
	Homogenization or sonication methods	Sonication (Sonic Vibracell TM), 5s x 5 times, 60% power
	Centrifugation	30 min / 2000xg
	Protein Precipitation	10% TCA added 1/1 v/v to sample, 1 h incubation, wash with 1 mL acetone
2.2.	Digestion
2.2.1	Pretreatment
	Reduction	2.5 mM DTT, 30 min, 56oC
	Alkykation	7 mM IAA, 30 min, 20oC
	Others	-
2.2.2	Digestion parameters
	Protein concentration	1 mg/mL
	Buffer	1.8 M urea, 15 mM Tris oH 8.5, phosphatase inhibitors, 2mM DTT, 25 mM ammonium bicarbonate
	Enzyme	Trypsin (Sigma)
	Incubation parameters	24 h
2.4.	Other treatments
	Description	Sample desalting with tC18 cartriges
4.	Peptide Purification/Fractionation
4.1.	HPLC (from MIAPE:Column Chromatography vs 1.0, June 2008)
4.1.1	Equipment
4.1.1.1	Product details for column
	Make	Hewlett Packard
	Model	Agilent 1100
	Separation mode	Strong Cation Exchange
4.1.1.2	Physical characteristics of column
	Length	200mm
	Diameter	2.1mm
	Description of stationary phase	Polysulfoethyl A,200 Angstrom
4.1.1.3	Equipment: Chromatography system used for separation
	Manufacturer and model	Agilent 1100 (binary pump, UV detector and manual injector)
4.1.2	Mobile phase
	Names of mobile phase	A and B
	Description of the constituents	A: mQ 0.1% formic (70%) ACN (30%),B: mQ 0.1% formic (70%) 500mM NH4Cl, ACN (30%)
4.1.3	Properties of the column run
	Time	80 min
	Gradient	0 min 0%B, 3 min 0%B,38 min 25%B,58min 100%B,78 min 100%B,80min 0%B
	Flow rate	200uL/min
	Temperature	20o C
	Separation purpose	Peptide Fractionation
4.1.4.1	Column outputs: Detection
	Equipment used for detection	UV Lamp
	Type	micro cell
	Equipment settings	220 nm
	Timescale over which data was collected	0 min to 80 min
4.1.4.2	Column outputs: Fractions
	Fraction name	SCX1_15 to SCX1_63
	Fraction description	Fractions were collected each 3 minutes from minute 15
5.	Phosphopeptide Enrichment
5.1.	Method 1
	Method description	IMAC
	Loading buffer	250 mM Acetic Acid / 30% ACN
	Resin	Phos-Select (Sigma)
	Incubation time	90 min / 20oC
	Washing buffer	250 mM Acetic Acid / 30% ACN
	Elution buffer	0.5% NH4OH (neutralized with formic acid)
	Other parameters	Evaporated to dryness and redisolved in 40 uL 0.1% FA for LC-MS
5.2.	Method 2
	Method description	TiO2
	Loading buffer	1 M glycolic acid, 5% TFA, 80% ACN
	Resin	Titansphere, GL Sciences, Japan
	Incubation time	-
	Washing buffer	1 M glycolic acid, 5% TFA, 80% ACN, 80% ACN 1% TFA
	Elution buffer	0.5% NH4OH
	Other parameters	Perform in a hand-made tip column. Evaporated to dryness and redisolved in 40 uL 0.1% FA for LC/MS

S3
Sample Preparation for Phosphoproteomics 2008/8/08
1	General features
1.1.	Date stamp	04/08/2008
1.2.	Responsible person or institutional role	Dra. Montserrat Carrascal. CSIC/UAB Proteomics Facility, IIBB-CSIC, Building M, Campus UAB, 08193 Bellaterra, Spain
1.3.	Sample name	Resting T-lymphocytes
2.	Sample processing
2.1.	Culture/tissue handling
2.1.1.	Tissue/Cell/Fluid type	T-lymphocytes
	Source	Buffy coat
	Species	Human
	Cell Isolation Method	Ficoll-Paque gradient centrifugation
	Cell/Pellet lysis	-
	Buffer	6 M urea, 50 mM Tris oH 8.5, phosphatase inhibitors, 2 mM DTT, 2 x 108 cell / mL
	Homogenization or sonication methods	Sonication (Sonic Vibracell TM), 5s x 5 times, 60% power
	Centrifugation	30 min / 2000xg
	Protein Precipitation	10% TCA added 1/1 v/v to sample, 1 h incubation, wash with 1 mL acetone
2.2.	Digestion
2.2.1	Pretreatment
	Reduction	2.5 mM DTT, 30 min, 56oC
	Alkykation	7 mM IAA, 30 min, 20oC
	Others	-
2.2.2	Digestion parameters
	Protein concentration	1 mg/mL
	Buffer	1.8 M urea, 15 mM Tris oH 8.5, phosphatase inhibitors, 2mM DTT, 25 mM ammonium bicarbonate
	Enzyme	Trypsin (Sigma)
	Incubation parameters	24 h
2.4.	Other treatments
	Description	Sample desalting with tC18 cartriges
4.	Peptide Purification/Fractionation
4.1.	HPLC (from MIAPE:Column Chromatography vs 1.0, June 2008)
4.1.1	Equipment
4.1.1.1	Product details for column
	Make	Hewlett Packard
	Model	Agilent 1100
	Separation mode	Strong Cation Exchange
4.1.1.2	Physical characteristics of column
	Length	200mm
	Diameter	2.1mm
	Description of stationary phase	Polysulfoethyl A,200 Angstrom
4.1.1.3	Equipment: Chromatography system used for separation
	Manufacturer and model	Agilent 1100 (binary pump, UV detector and manual injector)
4.1.2	Mobile phase
	Names of mobile phase	A and B
	Description of the constituents	A: mQ 0.1% formic (70%) ACN (30%),B: mQ 0.1% formic (70%) 500mM NH4Cl, ACN (30%)
4.1.3	Properties of the column run
	Time	80 min
	Gradient	0 min 0%B, 3 min 0%B,38 min 25%B,58min 100%B,78 min 100%B,80min 0%B
	Flow rate	200uL/min
	Temperature	20o C
	Separation purpose	Peptide Fractionation
4.1.4.1	Column outputs: Detection
	Equipment used for detection	UV Lamp
	Type	micro cell
	Equipment settings	220 nm
	Timescale over which data was collected	0 min to 80 min
4.1.4.2	Column outputs: Fractions
	Fraction name	SCX1_15 to SCX1_63
	Fraction description	Fractions were collected each 3 minutes from minute 15
5.	Phosphopeptide Enrichment
5.1.	Method 1
	Method description	TiO2
	Loading buffer	1 M glycolic acid, 5% TFA, 80% ACN
	Resin	Titansphere, GL Sciences, Japan
	Incubation time	-
	Washing buffer	1 M glycolic acid, 5% TFA, 80% ACN, 80% ACN 1% TFA
	Elution buffer	0.5% NH4OH
	Other parameters	Perform in a hand-made tip column. Evaporated to dryness and redisolved in 40 uL 0.1% FA for LC/MS

M1
HUPO-PSI MIAPE: Mass Spectrometry MS v2.24
1.	General features
1.a.	Global descriptors
1.a.1	Machine manufacturer, model	Lineal Ion Trap (LTQ, Thermo Fisher)
1.a.2	Estimated mass accuracy (ppm or Dalton) for all MS levels for which data are presented	0.3-0.6 Da
1.b.	Control and analysis software
1.b.1	Software name and version	Xcalibur 2.0
1.b.2	Switching criteria:	Dependent data analysis with neutral loss (MS and 8 MS/MS)
1.b.3	Isolation width	3 units (m/z)
2.	Ion sources
2.a.	Electrospray Ionisation (ESI)
2.a.1	Supply type	Chromatography
2.a.2	Scan cycle times	50 ms all levels (average)
2.a.3	Solvent flow rate and composition:	solvent A: Water 0.1% formic acid, solvent B: Acetonitrile 0.1% Formic Acid
2.a.4	Interface manufacturer, model	microESI from Thermo Fisher
2.a.5	Sprayer type, coating, manufacturer, model	Silica Tips, New Objective, FS360-100-30-CE-5
2.a.6	Relevant voltages where appropriate (tip,cone,acceleration)	Tubel lens = 130 V, cap lens = 48 V, spray voltage = 2 kV
2.a.7	Whether in-source dissociation performed:	No
3.	Post-source componentry
3.c.	Ion trap
3.c.1	Final MS exponent achieved	MS^3
3.d.	Collision cell
3.d.1	Gas type and pressure (bar)	unknow
3.d.2	Collision energy	0.35
3.f.	Detectors
3.f.1	Detector type	unknow
3.f.2	Detector sensitivity	unknow
4.	Peak list generation and annotation (a) Generation of spectrum
4.a.1	Location of source file including file name and type	Thermo RAW binary file generated by Xcalibur
4.a.2	Parameters triggering the generation of peak lists from raw data	Peak 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.3	Signal-to-noise estimation and method	ICIS 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.4	Smoothing; whether applied, parameters	ICIS algorithm: 15 smoothing points
4.a.5	Percentage peak height for centroiding; or algorithm used, if appropriate	ICIS algorithm
4.a.6	Background threshold, or algorithm used	ICIS algorithm: baseline window 40
4.a.7	Charge state calculation	Automatic charge state calculation: direct +1 direct assignement, +2/+3 alternatives redundantly generated
4.a.8	Ion mode for this spectrum	Positive

I1
HUPO-PSI MIAPE: Mass Spectrometry Informatics MSI v1.1
1	General features: a) Global descriptors
1.1.1	Software name, version and manufacturer	Bioworks 3.3, Thermo Electron Corp.
1.1.2	Customisations	"In house" software has been used for calculating the D-value (Peptide Prophet discriminant score ), filtering peptide results, obtain complementary information (MS state, retention time, spectra) and calculating Ascore (Gygi's group score to assign phosphorylation site).
1.1.3	Availability of the software	www.thermo.com/bioworks
1.1.4	Location of the files generated	Only filtered results are published at www.lymphos.org. Raw files and search results not publically available, but referenced for each spectrum (file name and scan number).
1.1.5	Description of Controls employed	Target-decoy database strategy employed.
2.	Input data and parameters
2.1.	Input data
2.1.1	Type of spectra	Raw Thermo files (*.RAW)
2.1.2	Description of MS data	Binary format.
2.1.3	MS data format	Temporary text files are generated by extract_ms program integrated in Bioworks suite
2.2.	Input parameters
2.2.1	Database queried	UniProt Knowledgebase Release 14.0. Swissprot/Trembl Human. 72595 proteins
2.2.2	Taxonomical restrictions	Human
2.2.3	Description of tool and scoring scheme	Target-decoy database. Reversed database appended to the end of the original database.
2.2.4	Specified cleavage agent(s)	Trypsin (KR_noP)
2.2.5	Allowed number of missed cleavages	2 missed cleavages
2.2.6	Additional parameters related to cleavage	Fully enzymatic: cleaves at both ends
2.2.7	Permissible amino acids modifications	Fixed: 57.0215 C Variable:15.994915 M / 79.966331 STY / -17.994915 ST
2.2.8	Precursor-ion and fragment-ion mass tolerance for tandem MS	Precursor:2 amu Fragment:0.8 amu
2.2.9	Thresholds; minimum scores for peptides, proteins	1% of False Positive Rate
2.2.10	Any other relevant parameters	Monoisotopic precursors and fragments. Ion series calculated for scoring: y and b.
4.	Interpretation and validation
4.1.1	Assessment and confidence given to the identification and quantitation	Peptide results are filtered using 1% of False Positive Rate, using XCorr and D-value (discriminant score used by Peptide Prophet). No protein related scores have been used, reporting all possible proteins inside used database for each peptide. Ascore has been used to assign phosphorilation site and provide an stimation of assignement confidence (high confidence ->90%- assignements present an Ascore > 15).
4.1.2	Results of statistical analysis or determination of false positive rate in case of large scale experiments	1% of False Positive Rate
4.1.3	Inclusion/exclusion of the output of the software are provided	Output data stored in relational database accessible in www.lymphos.org

MIAPE 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 and Mass Spectrometry Informatics 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
1	General features
1.1	Date stamp (as yyyy-mm-dd)	The date on which the work described was initiated; given in the standard yyyy-mm-dd format (with hyphens)
1.2	Responsible person or institutional role	The (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.
1.3	Sample/s name/s	Name of the samples processed. Include if there are control, standard of test samples.
2.	Sample processing
2.1.	Culture/tissue handling
2.1.1	Tissue/Cell/Fluid type	Biological material used in the experiment.
	Source	Source from which the material is obtained (p.e. T-cells from buffy coat)
	Species	Species of procedence
2.1.2	Tissue Isolation	Surgical procedure, laser capture, etc
2.1.2	Cell Isolation Method	Method used for cell isolation (e.g. Ficoll-Paque T-cells purification from buffy coat, enzymatic digestion, explant culture...)
2.1.3	Culture parameters
	Culture medium	Growth medium with all the complements added
	Culture conditions	Temperature and gas mixture (typically for mammalian cells, 37oC, 5% CO2)
	Cell density	Number of cells per mL
	Type of culture	Indicate if there are suspension or adherent cells and if they are primary cultures
	Time of culture	Time which the cells are in culture and number of previous passages
2.1.4	Cell/tissue activation
	Cell Treatment	Indicate cell treatment (activator,drug), including concentration and buffers used for addition
	Cell density	Number of cells per mL during treatment
	Time	Time for which the cells/tissues are treated.
2.1.5	Pellet preparation	Procedure used to prepare the pellet (wash, centrifugation or other parameters)
2.1.6	Cell/Pellet lysis
	Buffer	Buffer used for cell lysis. Indicate all the components, concentration and volume used for lysis
	Homogenization or sonication methods	Method used for cell/tissue disruption, include the time and power of the homogenization or sonication process.
	Centrifugation	Centrifugation time and centrifugal force
	Protein Precipitation	The method of precipitation if required. Include reagents, volumes and time of incubation
2.2.	Digestion
2.2.1	Pretreatment
	Reduction	Include reagents, time, temperature and concentration.
	Alkykation	Include reagents, time, temperature and concentration.
	Others	Other treatments before digestion. Include reagents, time, temperature and concentration.
2.2.2	Digestion parameters
	Protein concentration	Concentration of protein extract
	Buffer	Buffer used for digestion, indicating components, concentration and pH
	Enzyme	Enzyme description and concentration
	Incubation parameters	Incubation time and temperature
2.3.	Peptide/Protein Labeling
	Type of labeling	Type and make of the labeling reactive (e.g. iTRAQ from Applied Biosystems)
	Significative customizations	Any 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)
3.1.1	Gel matrix and electrophoresis
	Description of gel matrix	Gel 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 manufacturer	If 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 dimensions	The 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 distribution	As 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 concentration	The 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 ratio	The acrylamide to bisacrylamide ratio of the gel (Name of crosslinker and ratio described as "acrylamide:crosslinker")
	Gel lane	The number of lanes on the gel matrix
	sample application	Description 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
	Buffer	Description of the running buffers used, in terms of name of buffer, components with concentrations.
	Equipment	Apparatus used for electrophoresis. Include brand and model.
	Electrophoresis conditions	The running conditions applied to the gel (To be given in terms of voltages versus time/kilovolt hours, (or appropriate measurements) and temperature).
3.1.2	Staining	Staining method used to develop the gel image.
3.1.3	In gel digestion
	Enzyme	Enzyme description and concentration
	Incubation parameters	Incubation time and temperature
	Previous treatment	Previous treatment if required (e.g. Reduction and/or alkylation)
	Automatic digestor	If 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)
3.2.1	Equipment
3.2.1.1	Product details for column
	Make	The name of the manufacturer
	Model	The model number provided by the manufacturer
	Separation mode	A description of the type of column being used (e.g., Separation mechanism: affinity, anion exchange, cation exchange, reverse phase, size).
3.2.1.2	Physical characteristics of column
	Length	The length of the column.
	Diameter	The internal diameter of the column.
	Description of stationary phase	A description of the constituents of the stationary phase, including the name of the packing material and the particle size.
3.2.1.3	Chromatography system used for separation, where applicable	The name of the manufacturer and the model name provided by the manufacturer.
3.2.1.4	Mobile phase: for each mobile phase
	Name of mobile phase	Name used to refer to mobile phase in Properties of Column run
	Description of the constituents	For each constituent, a description and the concentration
3.2.1.5	Properties of the column run
	Time	The length of time for which the column is run. The value can be provided as a duration of time.
	Gradient	The 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 rate	The 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.
	Temperature	The temperature at which the column is run, including the period for which this holds if it varies during the experiment
	Separation purpose	Analytical or preparative
3.2.1.6	Column outputs - (a) detection
	Equipment used for detection	Make, model and description
	Type	A 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 settings	A description of control properties of the detector, such as the wavelength that is being detected
	Timescale over which data was collected	The time range covered by the trace produced by the detector
	Trace	The location and format of the trace
3.2.1.7	Column outputs - (b) fractions (if required)
	Fraction name	An optional name, unique within a run, by which a fraction can be referenced.
	Fraction description	Either 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)
3.2.2	Liquid digestion of fractions, if required	Indicate the enzyme used, time of digestion and concentration of sample and enzyme.
	Enzyme	Enzyme description and concentration
	Incubation parameters	Incubation time and temperature
	Previous treatment	Previous treatment if requires (e.g. Reduction and/or alkylation)
3.2.3	Other parameters	Other 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)
4.1.1	Equipment
4.1.1.1	Product details for column
	Make	The name of the manufacturer
	Model	The model number provided by the manufacturer
	Separation mode	A description of the type of column being used (e.g., Separation mechanism: affinity, anion exchange, cation exchange, reverse phase, size).
4.1.1.2	Physical characteristics of column
	Length	The length of the column.
	Diameter	The internal diameter of the column.
	Description of stationary phase	A description of the constituents of the stationary phase, including the name of the packing material and the particle size.
4.1.1.3	Chromatography system used for separation	The name of the manufacturer and the model name provided by the manufacturer.
4.1.2	Mobile phase: for each mobile phase
	Name of mobile phase	Name used to refer to mobile phase in Properties of Column run
	Description of the constituents	For each constituent, a description and the concentration
4.1.3	Properties of the column run
	Time	The length of time for which the column is run. The value can be provided as a duration of time.
	Gradient	The 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 rate	The 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.
	Temperature	The temperature at which the column is run, including the period for which this holds if it varies during the experiment
	Separation purpose	Analytical or preparative
4.1.4.1	Column outputs - (a) detection
	Equipment used for detection	Make, model and description
	Type	A 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 settings	A description of control properties of the detector, such as the wavelength that is being detected
	Timescale over which data was collected	The time range covered by the trace produced by the detector
	Trace	The location and format of the trace
4.1.4.2	Column outputs - (b) fractions (if required)
	Fraction name	An optional name, unique within a run, by which a fraction can be referenced.
	Fraction description	Either 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)
4.1.2	Other parameters	Other parameters not included in 4.1.1.
5.	Phosphopeptide Enrichment
5.1.	Method 1
5.1.1	Method description	Brief description of the method (e.g. IMAC purification)
5.1.2	Loading buffer	Buffer (type and volume) used to dissolve the sample and to load it into the resin
5.1.3	Resin	Material used for phosphopeptide enrichment. Include name of the manufacturer and reference.
5.1.4	Incubation time	Incubation time of the sample with the resin
5.1.5	Washing buffer	Buffer (type and volume) used to wash the resin after incubation.
5.1.6	Elution buffer	Buffer (type and volume) used to elute the phosphopeptides from the resin.
5.1.7	Other parameters	Other parameters not included.