The Association of Biomolecular Resource Facilities Proteomics Standards Research Group (sPRG) 2022 Multi-Species Standard to Assist Quantitative Proteomics

ABSTRACT: Authors: Benjamin A. Neely Josue Baeza Magnus Palmblad Lindsay K. Pino Brian C. Searle Susan T. Weintraub Study Participants The Proteomics Standards Research Group (sPRG) has record of creating successful materials and standards for the proteomics community. With the increased capacity and diversity of platforms, these materials have increased in complexity from the two synthetic peptides in the 2003 standard to the 147 synthetic phosphopeptides in the 2018 standard. The new standard builds on the rationale of a multi-species mixture for benchmarking label-free quantification and is composed of three proteome digests (over 10 000 proteins per species) in different ratios, where a complete detection by current techniques is unlikely. The materials were freeze-dried human, cow, and fish standards from NIST that are available for purchase. This new sPRG material may be used for evaluating systems and quantification workflows. Human liver, cow liver, and lake trout filet were purchased from NIST (RM 8461, SRM 1577c, and SRM 1947, respectively), and 8 to 12 mg of each was digested with TPCK-treated Worthington trypsin using S-Trap midi columns (ProtiFi). Resulting peptides were mixed at different levels across three aliquots, dried down, and distributed to participants for testing. Homogeneity and stability were evaluated within the sPRG; testing of data of sPRG members demonstrated the utility of the material when employing different label-free quantification methods. Sample sets were then disseminated to interested researchers across the globe to run in their own labs, with the goal of comparing the performance of the standard across different instrumentation, quantitative acquisition approaches and data processing workflows and software. The raw data from 13 participating labs are included here where the filename pattern is "ID_instrument_data type_sample_modifier", where sample is A, B or C premixed samples, and the modifier can be things like short_gradient or other user given information. Suggested fasta as well as verbose directions to participants is also given (please consult this). The participant data acquisition methods, reported ratios and additional analysis will be presented in a paper currently in preparation. Preliminary results from participant-submitted data showed two general observations. The reported species ratios were generally within the expected species ratios. Additionally, post data analysis showed that some peptides used to determine species ratios were shared between species. After filtering out the shared species peptides, the new species ratios showed greater accuracy and precision. The preliminary results suggest this multi-species mixture, and the data generated from it, are suitable for comparing analysis workflows with respect to protein inference and quantitative accuracy. This sPRG standard is the first step to a commercially available standard for benchmarking label-free proteomics platforms and workflows.

INSTRUMENT(S): Orbitrap Eclipse, Orbitrap Fusion Lumos, timsTOF Pro, Orbitrap Fusion, timsTOF HT, Orbitrap Exploris 480, Q Exactive HF, Q Exactive

ORGANISM(S): Bos Taurus (ncbitaxon:9913) Salvelinus Namaycush (ncbitaxon:8040) Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Benjamin Neely  

PROVIDER: MSV000097233 | MassIVE | Tue Mar 04 07:04:00 GMT 2025

SECONDARY ACCESSION(S): PXD061455

REPOSITORIES: MassIVE