N-Glycoprotein SRMAtlas

Abstract

Protein biomarkers have the potential to transform medicine as they are clinically used to diagnose diseases, stratify patients, and follow disease states. Even though a large number of potential biomarkers have been proposed over the past few years, almost none of them have been implemented so far in the clinic. One of the reasons for this limited success is the lack of technologies to validate proposed biomarker candidates in larger patient cohorts. This limitation could be alleviated by the use of antibody-independent validation methods such as selected reaction monitoring (SRM). Similar to measurements based on affinity reagents, SRM-based targeted mass spectrometry also requires the generation of definitive assays for each targeted analyte. Here, we present a library of SRM assays for 5568 N-glycosites enabling the multiplexed evaluation of clinically relevant N-glycoproteins as biomarker candidates. We demonstrate that this resource can be utilized to select SRM assay sets for cancer-associated N-glycoproteins for their subsequent multiplexed and consistent quantification in 120 human plasma samples. We show that N-glycoproteins spanning 5 orders of magnitude in abundance can be quantified and that previously reported abundance differences in various cancer types can be recapitulated. Together, the established N-glycoprotein SRMAtlas resource facilitates parallel, efficient, consistent, and sensitive evaluation of proposed biomarker candidates in large clinical sample cohorts. Molecular &

Applications of targeted proteomics in systems biology and translational medicine

“…Zawadzka and coworkers used both targeted and discovery proteomics to quantify a set of approximately 60 phosphopeptides from healthy human plasma following offline chromatography and immobilized metal ion affinity chromatography for phosphopeptide enrichment 94. Further advances in targeted proteomics of N‐glycosites will undoubtedly bring the N‐glyco Atlas providing SRM assays for 5568 N‐to‐D‐modified peptide sequences 95. These SRM‐assays were applied to prostate cancer tissue samples to determine aggressiveness of tumors using targeted extraction of peptide sequences from SWATH‐MS maps 96.…”

Using PeptideAtlas, SRMAtlas, and PASSEL: Comprehensive Resources for Discovery and Targeted Proteomics

“…The assays can be readily downloaded into a vendor specific acquisition method for the direct deployment in the next SRM experiment. At present SRMAtlas provides builds for the entire yeast (Picotti et al, 2013) and Mtb (Schubert et al, 2013) proteome, assays to target specific human and murine N-glyco-proteins (Hüttenhain et al, 2013), and a partial human and murine build mainly constructed from public available ion-trap data are available. SRMAtlas whole proteome builds for human, murine, bovine, porcine and rabbit with quadruple derived assays will be available here.…”

Targeted proteomics: a bridge between discovery and validation

“…This pipeline requires the development of assays that are sensitive, reproducible, quantitative and high throughput enough that a statistically significant evaluation can be made concerning the clinical value of each target [6–10]. To address these needs, assays have been developed using a technique known as selected reaction monitoring (SRM), which is a mass spectrometry technique that has recently been applied to the evaluation of quantitative differences between biological samples [5,11,12,16–18]. …”

“…To address the first hurdle, there is need for sample preparation and automation [53]. To address the second hurdle, publically available tools are available, such as PASSEL, NIST peptide library and SRMAtlas, which list spectral libraries, which can be used to develop SRM assays [5,54,55]. However, there is still a need for accurately predicting the fragmentation patterns and collision energies that will yield the most sensitivity in a triple quad instrument.…”

“…There is a growing consensus among leaders in the field that the development of quantitative high-throughput assays for the candidate proteins represents a bottleneck between biomarker discovery and fully validated assays for use in the clinic [4–10]. Targeted proteomics affords the potential for the development of assays that are sensitive, reproducible, quantitative and possess a high enough throughput that statistically relevant conclusions can be drawn about the potential clinical value of a biomarker [5,11–15]. …”