Manual method of visually identifying candidate signals for a targeted peptide.

Journal of Chromatography B

Aleksey D. Filimonov *ORCID iD icon, Arthur T. Kopylov , Andrey V. Lisitsa, Alexander I. Archakov

Last Updated April 21, 2019

Supplementary materials

The supplementary materials for the article do not include s2pe reports used to accomplish experiments with the standard manual method of visually Identifying Candidate Signals for a targeted Peptide (ICSP): The reader can build these reports using srm2prot Express software (s2pe) as described in section 3 of the Supplement (MS Word 2010). However, for the reader's convenience, the s2pe reports we built and analyzed for the study are presented in this page with the most-used s2pe-report features. In addition, the unpacked and expanded supplementary materials are also available here.

We applied the ICSP method to re-interpret our LC-SRM data series ds0. The original and transformed XIC groups were built from ds0 by s2pe. Use of the ICSP method revealed the mistakes we made when previously interpreting ds0 using the conventional technique improved by statistical analysis (see section 2 of the article and section 1.3 of the Supplement). s2pe reports contain original and transformed XIC groups with identified peptide candidate signals. These reports are available for browsing as individual MS Excel documents (613 MB in total) and for downloading as one archive (431M) in 7-Zip format.

To browse s2pe reports in MS Excel (2007 or later), use s2pe Navigator (the MS Excel Add-in), which is a part of srm2prot Express software and can be installed in 3 clicks. After downloading and unpacking the archive of s2pe reports, open the MS Excel worksheet ds0.xlsx, the catalog of the peptide candidate signals identified in ds0. The catalog contains hyperlinks to all the s2pe reports and identified signals with their properties such as widths, maxima and squares.

Catalog of the peptide candidate signals identified in ds0

An sp2e report contains four sheets: Assay, Data, Chromatograms and Signals. For one run represented by one acquired raw data file, the sheet "Chromatograms" displays the XIC group in four special views. If signals were identified, they are also shown in dash-dot frames. The signal's color reflects the signal's status: "target" in green, "candidate" in orange and "invalid" in blue.

XIC group with the signal of
light endogenous peptide GWVTDGFSSLK of protein APOC3_HUMAN acquired from Biognosys AG’s PlasmaDive Kit™

The sheet "Signals" displays all the signals identified in the XIC group. The maximum of and square under a corresponding signal's XIC are calculated and updated automatically during input. The sub-header row enumerates corresponding signal's XICs. Use the column "note" to provide additional information for a signal. To make the signal's endpoints more precise, use columns "from, s" and "to, s". s2pe Navigator supports sorting signals with standard Excel's functions.

Sheet "Signals" of s2pe report P00167.YYTLEEIQK.r-HLV_B-r001.xlsx

The sheet "Assay" reports the details of the XIC group, the acquisition method, the run and the processing method. The respective tables map precursor and product ions to the corresponding channels and XICs, displaying given m/z values and accurate m/z values of ions. The m/z-window width options used to acquire the XIC group presented in the s2pe report are displayed in columns "Q1 res" and "Q3 res".

Assay prototype (some features are under implementation)

Unpacked and expanded supplementary materials

The Supplement (MS Word 2010) contains a brief overview about SRM software for chromatogram interpretation, a short description of ms2prot methods and features in comparison with mProphet, the use cases and features of s2pe and the instruction for processing our SRM data series ds0.
The MS Excel worksheet ICSP_ds0_stat.xlsx contains the summaries and distributions for XIC groups built from ds0 and for identified peptide candidate signals.
The MS Excel worksheet ds0_jpr2012 vs ICSP.xlsx contains the source data and comparison results presented in the article.
The database cmp_v_jpr2012.accdb (760K, "as is", MS Access 2007) was built to compare the peptide signals identified previously (using the conventional signal identification technique with MassHunter+ms2prot+mProphet+PASSEL) and the peptide candidate signals identified in this study (using the ICSP method).

Browse the s2pe reports from ds0 as individual MS Excel documents in the web directory
The s2pe report P02766.GSPAINVAVHVFR.r-mix_E_dmrm-r005.xlsx illustrates case 5 of Table 2.

Configurations for and logs of building the s2pe reports from ds0
File Description
subst.tsv The label substitution table used to rename XIC groups from ds0; tab-separated values.
ds0.cfg The configuration file used to process ds0 with s2pe; plain text.
192_1-QC-A6A8BOQ.xml The acquisition method with corrected parameters for protein A6A8BOQ.
all-0.lst The copy of a file list given to s2pe for processing ds0.
all.log The text log written by s2pe while processing ds0.
ds0.log The text log written by utility mk_sig_cat of s2pe while building the catalog of peptide candidate signals identified in ds0.

The performance of s2pe can be estimated by the following screenshots obtained from Sysinternals’s Process Explorer while processing ds0 on a system with Intel® Core™ i5-3450 CPU 3.50 GHz and MS Excel 2010.

Runtime properties of the s2pe process while building reports
Performance graph of the s2pe process while building reports
Runtime properties of the Excel process importing the catalog of the peptide candidate signals while s2pe processed ds0
Performance graph of the Excel process importing the catalog of the peptide candidate signals while s2pe processed ds0
Runtime properties of the Excel process while building the catalog of the peptide candidate signals identified in ds0
Performance graph of the Excel process while building the catalog of the peptide candidate signals identified in ds0

The web directory of the supplementary materials was prepared in 2015 by Aleksey Filimonov for Orekhovich IBMC RAS and the article.