Cross-platform Clinical Proteomics using the Charité Open Standard for Plasma Proteomics (OSPP)
Description
we present the Charité Open Peptide Standard for plasma proteomics (OSPP), an open resource composed of 211 isotope-labeled peptides, intended to be used as an internal standard for plasma and serum proteomic projects. The OSPP was designed based on peptides consistently quantified across a panel of diverse human studies, is made of peptides that can easily be synthesized, that distribute equally over chromatographic gradients, and that show consistent identification performance across diverse LC-MS platforms, acquisition methods, and matrices. raw proteomics data have been deposited in the ProteomeXchange under accession code PXD070765
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Raw Data Processing All raw data from the ZenoTOF 7600 system was acquired by SCIEX OS (v. 3.0). All raw data from timsTOF HT were acquired with timsControl (v.5.1.8) and HyStar (v.6.3.1.8). All raw data from Exploris 480 (Thermo) were acquired using Xcalibur. Discovery proteomics The raw proteomics data from all DIA methods was processed using DIA‐NN, 1.8.1, available on GitHub (DIA‐NN GitHub repository 78). The MS2 and MS1 mass accuracies were set to 20 and 12 ppm (ZenoTOF 7600 data) or 15 and 15 ppm (timsTOF and Exploris 480 data), and the scan window to 7. The aforementioned OSPP-specific Human Spectral Library is used for data processing with additional commands: --fixed-mod SILAC,0.0,KR,label --channels SILAC,L,KR,0:0; SILAC,H,KR,8.014199:10.008269 --peak-translation --original-mods --matrix-ch-qvalue 0.01 --restrict-fr --report-lib-info Specifically, following a two-step MBR approach 28, an in silico spectral library is first generated by DIA-NN from the FASTA file(s); this library is then refined based on the DIA dataset and subsequently used to reanalyze the dataset to obtain the final results. The data were filtered in the following way. First, a 1% run-specific q-value filter per isotope channel was automatically applied at the precursor level by DIA-NN (--matrix-ch-qvalue 0.01). We note that in any experiment processed using the MBR mode in DIA-NN, 1% global precursor q-value filtering is also applied automatically 28. For quantification, we used the “Precursor.Translated” value as quantities for each precursor in MS2 quantification. For Exploris 480 data, since orbitraps are sensitive in MS1, we also used “Ms1.Translated” was used. Targeted proteomics Zeno MRM-HR data were processed using Skyline (64-bit, v.23.1.0.268). No blinding was performed during peak integration. The relative quantity of each peptide is calculated by the summation of peak areas of each selected fragments of a peptide (fragments used in DIA calculation extracted from DIA-NN output
Institutions
- Charite Universitatsmedizin Berlin