MSACL 2016 US Abstract

Design of a LC-MS/MS Plasma Protein Assay Using Data Independent Acquisition

Jarrett Egertson (Presenter)
University of Washington

Bio: Dr. Jarrett Egertson is the services lead for the MacCoss lab at the University of Washington. He earned a B.S. in Molecular, Cellular, and Developmental Biology at the University of California, Los Angeles, and a Ph.D. in Genome Sciences at the University of Washington. He has been developing novel DIA approaches in the MacCoss lab since 2008 with a focus on improving precursor selectivity via spectral multiplexing and developing practical software solutions for DIA data analysis.

Authorship: Jarrett D. Egertson (1), James G. Bollinger (1), Ying S. Ting (1), Clark M. Henderson (2), Vagisha Sharma (1), Andrew N. Hoofnagle (2), Michael J. MacCoss (1)
(1) Department of Genome Sciences, University of Washington School of Medicine, Seattle, WA, USA. (2) Department of Lab Medicine, University of Washington School of Medicine, Seattle, WA, USA

Short Abstract

A data independent acquisition (DIA) approach is used to generate a highly multiplexed plasma protein assay. Using DIA, we build a chromatogram library containing relative fragment ion abundances and retention times for thousands of peptides detected in human plasma. Significantly, we use the high multiplexing capacity of DIA to expedite assay validation including assessment of digestion kinetics, peptide stability, and quantitative linearity. Additionally, we demonstrate an external calibration approach for DIA-based assays using a pooled plasma standard. The plasma standard is measured intermittently using a single LC-MS/MS DIA injection. The signal for each peptide measurement can then be calibrated to the signal for the same peptide in the plasma standard to control for batch effects and normalize to a common reference.

Long Abstract


We demonstrate the application of data independent acquisition (DIA) at multiple stages of building a multiplexed, LC-MS/MS based plasma protein assay. Similar to selected reaction monitoring (SRM) approaches, data independent acquisition (DIA) approaches quantify analytes based on the signal of a set of selective fragment ions measured by MS/MS and integrated over time. Unlike SRM, DIA approaches comprehensively measure MS/MS data for every precursor in a wide m/z range (e.g. 500 – 900 m/z) by acquiring a repeated cycle of wide isolation window MS/MS scans using a full scan mass analyzer. With the DIA approach, MS/MS data for quantitation can be extracted for any query peptide within a wide precursor m/z range thus enabling highly multiplexed assays at the expense of selectivity compared to SRM. We demonstrate how the multiplexing capacity of DIA can be used to expedite numerous steps in the development of a plasma protein assay. Specifically we use DIA to assess peptide MS/MS signal, chromatography, retention time, digestion kinetics, stability, and quantitative linearity. Additionally, we demonstrate an external calibration approach for DIA-based assays using a pooled plasma standard.

Chromatogram Library Generation:

To determine which peptides are detectable in plasma, DIA data is acquired on a pooled plasma standard using ten injections which collectively measure peptide precursors between 400 and 1000 m/z. To obtain maximal sensitivity in this step, each injection measures a 50 m/z precursor range with a repeated cycle of a 50 m/z wide MS scan and 25 adjacent 2 m/z wide MS/MS scans. An in-house search tool (Pecan, manuscript in preparation) is used to detect peptides in these analyses using a peptide-centric approach (1). The extracted MS/MS chromatograms and normalized retention time for each detected peptide is stored in a chromatogram library. Normalized retention times are calculated relative to 15 isotopically-labeled peptide retention time calibration standard (Thermo PRTC mix) spiked-in post-digestion. This step of the analysis resulted in a chromatogram library containing 4,244 unique fully-tryptic peptides.

Assessment of Peptide Stability:

The stability of each peptide in the chromatogram library was assessed by analyzing the same plasma digest (10 DIA runs) after incubating the digested sample at 4°C for 3 days. Unstable peptides showed a substantially reduced intensity post-incubation compared to pre-incubation.

Digestion Kinetics:

A digestion time-course was acquired on the plasma standard to measure digestion kinetics for plasma peptides. Data were acquired on the plasma standard digested for 0.25, 1, 2, 4, 10, and 18 hours using a single DIA LC-MS/MS run for each time point. From these data, the digestion kinetics for a large subset of the total 4,244 peptides in the chromatogram library could be assessed and used to eliminate peptides with unstable kinetics at a particular digestion time.

Linearity Assessment:

To assess quantitative linearity of the DIA approach, the digested plasma standard was mixed with digested chicken plasma at ratios varying from 10:1, to 1:10 (2). These mixtures were each measured using single injection DIA LC-MS/MS runs (20 x 20 m/z windows covering 500 – 900 m/z) acquired in triplicate.

External Calibration:

A commercially-available, human plasma standard consisting of pooled plasma from 200 males and 200 females (Golden West Biologicals; Temecula, CA) is processed in each batch of sample digestions and measured by a single injection DIA LC-MS/MS run every 10 sample injections. Extracted peptide signal intensity from these standard runs are used to normalize the signal from sample injections. This process normalizes for batch processing effects and makes all measurements relative to a common reference standard to provide for better comparison between experiments (3).


The multiplexing ability of DIA is useful beyond extending the breadth of an LC-MS/MS assay. It can be applied to assess the MS/MS signal, chromatography, retention time, digestion kinetics, stability, and quantitative linearity of peptides in the assay using less injections and instrument time than would be required to make these characterizations for a large number of peptides by traditional targeted approaches.

References & Acknowledgements:

(1) Ting YS, et. al., Mol. Cell. Proteomics 2015

(2) Grant RP, Hoofnagle AN, Clinical Chemistry 2014

(3) Cox HD, Lopes F, et. al., Clinical Chemistry 2014

Financial Disclosure

GrantsyesThermoFisher Scientific
Board Memberno

IP Royalty: no

Planning to mention or discuss specific products or technology of the company(ies) listed above: