Mass spectrometric protein maps for biomarker discovery and clinical research

Abstract

Among the wide range of proteomic technologies, targeted mass spectrometry (MS) has shown great potential for biomarker studies. To extend the degree of multiplexing achieved by selected reaction monitoring (SRM), we recently developed SWATH MS. SWATH MS is a variant of the emerging class of data-independent acquisition (DIA) methods and essentially converts the molecules in a physical sample into perpetually re-usable digital maps. The thus generated SWATH maps are then mined using a targeted data extraction strategy, allowing us to profile disease-related proteomes at a high degree of reproducibility. The successful application of both SRM and SWATH MS requires the a priori generation of reference spectral maps that provide coordinates for quantification. Herein, we demonstrate that the application of the mass spectrometric reference maps and the acquisition of personalized SWATH maps hold a particular promise for accelerating the current process of biomarker discovery.

Keywords::

• biomarker discovery
• diagnostics
• digital maps
• mass spectrometry
• proteomics
• reference maps
• selected reaction monitoring
• SWATH MS

Acknowledgment

We would like to acknowledge C Raison for editorial assistance and the whole Aebersold group for the discussion about biomarker discovery projects that led directly to the considerations of the issues discussed in this manuscript.

Financial & competing interests disclosure

Research in the group is supported by early access to advanced instrumentation from AB/Sciex under a collaborative research agreement. The authors acknowledge the support from the National Institutes of Health (NIH Grant # U01CA152813). The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

Key issues

• • The clinical needs of personalized medicine and unbiased biomarker discovery drive the high-throughput, multiplexed, quantitative and cost-effective investigation of clinical proteomes. The better annotation of these proteomes benefits greatly from mass spectrometric reference maps.

• • Mass spectrometric reference maps consisting of assays for targeted quantification of PTPs are established or under development such as those of plasma proteome, N-linked glycoproteome, CAPs sets and even the human global proteome.

• • The mass spectrometric reference maps are generated by either deep shotgun sequencing of biological samples or by chemically synthesizing a suitable set of peptides that uniquely represent each component in the system. All assays are made publicly accessible through websites such as PeptideAtlas and SRMAtlas.

• • Traditional antibody-based protein measurements are the method of choice if a validated assay has been developed. They suffer, however, from the high cost required for their development.

• • Shotgun proteomics-based validation, although enabling global protein identification and biomarker screening in unbiased way, mainly suffers from an attrition of proteins detected and quantified between samples.

• • SRM-based targeted analysis of proteins in complex samples offers stable, sensitive, quantitative and reproducible quantification. It achieves LOD in the low attmole range in different clinical samples and even 100–1000 lower by utilization of peptide antibody-based enrichment. The SRM-based biomarker discovery pipeline has been widely and successfully used in clinical proteomics. The pipeline is now significantly accelerated by mass spectrometric maps generated for clinical proteomes

• • SWATH MS technology was developed to address the problem of low analyte throughput in SRM analysis. As a DIA acquisition method, SWATH MS generates a complete recording of the fragment ion spectra of all analytes detectable in a clinical sample in 2–3 h. SWATH MS coupled with targeted data extraction achieved a slightly lower sensitivity, comparable accuracy, dynamic range and reproducibility compared to state-of-the-art SRM measurements, and therefore combines the strengths of shotgun and SRM analysis.

• • The digitalized SWATH maps generated by SWATH MS can be annotated using mass spectrometric reference maps to identify and quantify the global detectable proteome across samples. The SWATH map represents a fast, deep, reproducible and most importantly permanent proteomic recording which promises to avoid sample storage and to support the iterative biomarker discovery and personalized proteome phenotyping.

• • We propose that mass spectrometric reference maps and SWATH MS technology will be widely applied in clinical research and the SWATH digital map-assisted biomarker discovery pipeline will to some extent replace the current shotgun or SRM-based pipelines.

Notes