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

Journal of Proteome Research, vol. 25, pp. 4251–4261

Abstract

Mass spectrometry-based proteomics has advanced through parallel improvements in instrumentation (mass spectrometers) and software (data analysis), yet whether these improvements interact synergistically or provide diminishing returns remains unclear. Here, we systematically evaluate instrument-software coevolution across eight mass spectrometry platforms, three generations of search engines, and multiple rescoring approaches, yielding 72 unique instrument-software combinations spanning from 2004 to 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, thereby increasing the population of challenging spectra for which 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

   Ghent University · VIB-UGent Center for Medical Biotechnology

   Affiliation as printed

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

   Ghent University , , ,

2. Robbin Bouwmeester

   Ghent University · VIB-UGent Center for Medical Biotechnology

   Affiliation as printed

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

   Ghent University , , ,

3. Arthur Declercq

   Ghent University · VIB-UGent Center for Medical Biotechnology

   Affiliation as printed

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

   Ghent University , , ,

4. Robbe Devreese

   Ghent University · VIB-UGent Center for Medical Biotechnology

   Affiliation as printed

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

   Ghent University , , ,

5. Magnus Palmblad Aachen

   Leiden University Medical Center

   Affiliation as printed

   Leiden University Medical Center , , ,

6. Wout Bittremieux

   University of Antwerp

   Affiliation as printed

   University of Antwerp , , ,

7. Tess A. V. Afanasyeva

   Amsterdam University Medical Centers · University of Amsterdam

   Affiliation as printed

   Amsterdam UMC, University of Amsterdam , , ,

8. Joel Lapin

   Technical University of Munich

   Affiliation as printed

   Technical University of Munich , , ,

9. Arzu Tuğçe Güler

   Boston Children's Hospital · Northeastern University · Harvard University

   Affiliation as printed

   Boston Children’s Hospital , , , ,

   Harvard Medical School , , ,

   Northeastern University , , , ,

10. Pierre-Olivier Schmit

    Bruker (France)

    Affiliation as printed

    Bruker France SAS , ,

11. Christine Carapito

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

    Affiliation as printed

    IPHC UMR 7178, University of Strasbourg, CNRS , , ,

    Infrastructure Nationale de Protéomique ProFI − UAR 2048 , ,

12. Lennart Martens corresponding

    Centre National de la Recherche Scientifique · Institut Pluridisciplinaire Hubert Curien · Ghent University · VIB-UGent Center for Medical Biotechnology · Proteomics French Infrastructure Core · Université de Strasbourg

    Affiliation as printed

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

    Ghent University , , ,

    IPHC UMR 7178, University of Strasbourg, CNRS , , ,

    Infrastructure Nationale de Protéomique ProFI − UAR 2048 , ,

13. Ralf Gabriels

    Ghent University · VIB-UGent Center for Medical Biotechnology

    Affiliation as printed

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

    Ghent University , , ,