Classification
1.
2.
Definition
General features — Global descriptors of the experiment
1.1. Experiment identifier or name
SRM_1
1.2. Responsible person or role
Dra. Marina Gay. CSIC/UAB Proteomics Facility, IIBB-CSIC, Building M, Campus UAB, 08193
Bellaterra, Spain
1.3. Quantitative approach
Label-free pattern-based, SRM
Experimental design and sample description —2.1 Experimental design
2.1.1 Groups
3 human plasma spiked with two human proteins and two rabbit proteins at different
concentration (ABRF PRG2009 study)
2 experimental replicates
2.1.2 Biological and technical replicates
2.
Experimental design and sample description —2.2 Sample / Assay description
Labelling protocol (if applicable)
na
Sample description
The spiked proteins are: Prostate specific antigen (human), Beta chorionic gonadotropin
(human), Glycogen phosphorylase A (rabbit), Glycogen phosphorylase B (rabbit).
Spiked in a range of 2.5 fmol/uL to 1.25 pmol/uL
Sample name
A,B,C,D,E;F
2.2.2.2 Sample amount
1 mg before serum depletion and SCX fractionation.
2.2.2.3 Sample labelling with assay definition, i.e. MS run / data set together
with reporting ion mass, reagent or isotope labelled amino acid
na
2.2.2.4 Replicates and/or groups
A-E, B-F, C-D
2.2.3 Isotopic correction coefficients
na
2.2.4 Internal references
1 serum protein (CERU_HUMAN) to normalize the XIC areas
3.
4.
Input data — Description and reference of the dataset used for quantitative analysis: type, format and availability of the data. No actual values are requested here.
3.1. Input data type
Extracted ion chromatograms (SRM chromatograms)
3.2. Input data format
Thermo.raw files
3.3. Input data merging
4 SCX fractions
3.4. Availability of the input data
23 raw files
Protocol —Description of the software and methods applied in the quantitative analysis (including transformation functions, aggregation functions and statistical calculations).
4.1. Quantification software name, version and manufacturer
Xcalibur 2.1 (ThermoFischer)
4.2. Description of the selection and/or matching method of features,
together with the description of the method of the primary
extracted quantification values determination for each feature
m/z of the precursor and 2 or 3 fragment ions, and retention time for peak selection.
For quantitation the area in the extract ion chromatogram was used.
and/or peptide
4.3. Confidence filter of features or peptides prior to quantification
m/z with 0.5 Da error and retention time with 1 min window. Manual validation.
4.4. Description of data calculation and transformation methods
The area of the XIC for the different transitions was used and then normalized with the area
obtained from the internal reference.
4.4.1.
Missing values imputation and outliers removal
na.
4.4.2.
Quantification values calculation and / or ratio
determination from the primary extracted quantification
Average of sample replicates for each condition.
values
4.4.3.
Replicate aggregation
na
4.4.4.
Normalization
Data normalized using XIC areas of the internal reference
Protein quantification values calculation and / or ratio
na
4.4.5.
determination from the peptide quantification values
4.4.6.
5.
Protocol specific corrections
na
4.5. Description of methods for (statistical) estimation of correctness
PCA analysis to group samples
4.6. Calibration curves of standards
na
Resulting data —Provide the actual quantification values resulting from your quantification software together with their estimated confidence. Depending of the quantification technique
or even of the quantification software, only some of the following items could be satisfied (e.g., for spectral counting, only quantification values at protein level can be provided)
5.1 Quantification values at peptide and/or feature level: Actual quantification values achieved for each peptide and/or, in case of feature-based quantification, for the corresponding features
(mapped back from each peptide), together with their estimated confidence.
5.1.1 Primary extracted quantification values for each feature (e.g. area,
height, etc.), with their statistical estimation of correctness
http://proteo.cnb.csic.es/downloads/miape-quant/Results_peptides_SRM_LPCSICUAB.xlsx
5.1.2 Quantification values for each peptide as a result of the aggregation of
the values of the previous section (5.1.1), with their statistical estimation of
correctness
http://proteo.cnb.csic.es/downloads/miape-quant/Results_peptides_SRM_LPCSICUAB.xlsx
5.2 Quantification values at protein level: Actual quantification values achieved for each protein and for each protein ambiguity group, together with the confidence in the quantification
value.
Basic / raw quantification values with statistical estimation of correctness
http://proteo.cnb.csic.es/downloads/miape-quant/Results_proteins_SRM_LPCSICUAB.xlsx
5.2.2 Transformed / aggregated / combined quantification values of the
proteins at group level, with their statistical estimation of correctness
http://proteo.cnb.csic.es/downloads/miape-quant/Results_proteins_SRM_LPCSICUAB.xlsx