Instrument-Software Synergy in Proteomics: Systematic Evaluation Across Mass Spectrometry Platforms, Search Engines, and Rescoring Methods

Zenodo (CERN European Organization for Nuclear Research)

Abstract

Mass spectrometry-based proteomics has advanced through parallel improvements in instrumentation (mass spectrometer) and software (data analysis), yet whether these improvements interact synergistically or provide diminishing returns remains unclear. Here, we systematically evaluate instrument-software co-evolution across eight mass spectrometry platforms, three generations of search engines, and multiple rescoring approaches, yielding 72 unique instrument–software combinations spanning from 2004–2024. Our results reveal that instrumentation and software improvements produce synergistic rather than substitutive benefits. Here, machine learning-based rescoring consistently recovers identifications from low-intensity precursors that produce noisier, more challenging spectra. Crucially, because of increased sensitivity and speed, modern instruments detect more low-intensity precursors, increasing the population of challenging spectra where rescoring provides the greatest benefit. However, this expanded detection depth comes at a cost: recovered low-abundance peptides exhibit inherently higher quantification error, creating a fundamental trade-off between proteome coverage and quantification accuracy. Together, these findings provide a systematic overview of how instrument and software advances have jointly shaped proteomics performance over the past two decades.

Authors 13

1. Sander Heyndrickx

   VIB-UGent Center for Medical Biotechnology

   Affiliation as printed

   CompOmics, VIB-UGent Center for Medical Biotechnology, VIB, Ghent, Belgium

2. Ralf Gabriels

   VIB-UGent Center for Medical Biotechnology

   Affiliation as printed

   VIB-UGent Center for Medical Biotechnology

3. Robbin Bouwmeester

   Ghent University · VIB-UGent Center for Medical Biotechnology

   Affiliation as printed

   VIB-UGent & Janssen

   VIB UGent Centrum voor Medische Biotechnologie

4. Robbe Devreese

   VIB-UGent Center for Medical Biotechnology

   Affiliation as printed

   VIB-UGent Center for Medical Biotechnology

5. Arthur Declercq

   VIB-UGent Center for Medical Biotechnology

   Affiliation as printed

   VIB-UGent Center for Medical Biotechnology

6. Lennart Martens

   Ghent University · VIB-UGent Center for Medical Biotechnology

   Affiliation as printed

   VIB UGent Centrum voor Medische Biotechnologie

   Ghent University

7. Magnus Palmblad Aachen

   Leiden University Medical Center

   Affiliation as printed

   Leiden University Medical Center

8. Wout Bittremieux

   University of Antwerp · University of California San Diego

   Affiliation as printed

   University of California San Diego

   University of Antwerp

9. Tess A. V. Afanasyeva
10. Joel Lapin
11. Arzu Tuğçe Güler

    Northeastern University

    Affiliation as printed

    Northeastern University

12. Pierre-Olivier Schmit
13. Carapito Christine

    Centre National de la Recherche Scientifique · Institut Pluridisciplinaire Hubert Curien · Proteomics French Infrastructure Core · Université de Strasbourg

    Affiliation as printed

    Infrastructure Nationale de Protéomique ProFI − UAR 2048, Strasbourg, 67087, France

    BioOrganic Mass Spectrometry Laboratory (LSMBO), IPHC UMR 7178, University of Strasbourg, CNRS, Strasbourg, 67000, France