Classification
1.
2.
2.
Definition
General features — Global descriptors of the experiment
1.1. Experiment identifier or name
Proteomic Quantitative analysis of the Salmonella typhimurium RcsB regulon
1.2. Responsible person or role
Alberto Paradela/Rosana Navajas (Laboratorio de Proteómica-CNB-CSIC; telf.
915854540)
1.3. Quantitative approach
ICPL (Duplex version: ICPL-6C12 vs ICPL-6C13)
Experimental design and sample description —2.1 Experimental design
2.1.1.
Groups
ICPL1: Salmonella RcsB wild type (ICPL-6C12) vs. Salmonella RcsB-IgaA1
mutant (ICPL-6C13)
2.1.2.
Biological and technical replicates
No replicates were performed
Experimental design and sample description —2.2 Sample / Assay description
2.2.1 Labelling protocol (if applicable)
ICPL-labeling at peptide level; experimental protocol followed manufacturer´s
instructions.
Sample description
For each sample, provide, if applicable, the following information:
Sample name
ISM WT: Salmonella typhimurium grown in ISM medium and expressing an
unaltered RcsB regulon.
ISM IgaA: Salmonella typhimurium grown in ISM medium and expressing a
mutated RcsB regulon (mutation in IgaA1).
2.2.2.2 Sample amount
100 g per sample
ISM WT labelled with ICPL-6C12; ISM IgaA labelled with ICPL-6C13
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
ICPL-6C12: 105.0215 Da
ICPL-6C13: 111.0419 Da
m: 6.0204 Da
2.2.2.3 Replicates and/or groups
No replicates were performed
2.2.3 Isotopic correction coefficients
Not appliable
2.2.4 Internal references
Not appliable
3.
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
3.2. Input data format
4.
XIC (extracted ion chromatogram) obtained from the full MS scan.
.yep files (Bruker)
3.3. Input data merging
Sample was fractionated using basic pH-Reversed-phase chromatography,
collecting 30 fractions. Each fraction was individually analyzed by LC- ESI
MSMS. Datasets corresponding to each fraction were merged, creating a single
combined dataset, by the AnalysisCombiner tool included in the DataAnalysis
v.3.4. (Bruker Daltonics).
3.4. Availability of the input data
available as requested
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
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 and/or peptide
WARP-LC 1.1 (Bruker Daltonics)
-Mass tolerance and Retention time tolerance for isotopic substitution: 0.8 Da
& 40 sec.
-Extracted ions from 1-, 2- and 3-charged ions were used (smoothing for each
EIC is performed).
-Singlet is defined when ratio>50 or <0.02.
-Quantitation is performed by considering the height of the monoisotopic
peaks.
4.3. Confidence filter of features or peptides prior to quantification
Quantification applied to peptides below the threshold of FDR <5%
4.4. Description of data calculation and transformation methods
Provide, if applicable, the following information. Also state at which level
(feature, peptide and/or protein) are performed and in which order:
(mathematical formulas are allowed)
4.4.1.
4.4.2.
Missing values imputation and outliers removal
Manual evaluation of the outliers
Quantification values calculation and / or ratio determination from the
- Intensity values for each labeled peptide represent the mean of the intensity
values of the signals in retention time range specified in 4.2: 40 sec. Ratios are
calculated dividing the intensity of the signals corresponding to either the
peptide labeled with the light or heavy form of ICPL.
primary extracted quantification values
4.4.3.
Replicate aggregation
no replicas were performed
4.4.4.
Normalization
no normalization procedures at the peptide level were performed
4.4.5.
Protein quantification values calculation and / or ratio determination from
the peptide quantification values
4.4.6.
Protocol specific corrections
4.5. Description of methods for (statistical) estimation of correctness
4.6. Calibration curves of standards
-Protein quantification values are inferred after calculating the arithmetic
average of the quantification ratios found for the peptides belonging to that
protein.
-No correction for shared peptides was used.
- Only proteins identified with at least two peptides were considered for
quantification.
No specific corrections were performed
- WARP-LC calculates the standard deviation of the ratios for each protein.
Median of the protein ratios was calculated to correct (by substraction)
the systematic errors of the entire workflow.
no calibration curves were used
5.
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
5.1.2.
http://proteo.cnb.csic.es/downloads/miape-quant/ISM_WT vs IgaA stock1
5nov09_protein and peptide.xlsx
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
not applicable
correctness
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.
5.2.1 Basic / raw quantification values with statistical estimation of correctness
http://proteo.cnb.csic.es/downloads/miape-quant/ISM_WT vs IgaA stock1
5nov09_protein.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/ISM_WT vs IgaA stock1
5nov09_protein.xlsx