MSACL 2016 US Abstract

Getting the Biggest Bang for the Buck: Manipulating Trypsin Digestion Conditions to Accelerate Digestion and Improve Signal Intensity

Yu Zi (Emma) Zheng (Presenter)
St.Paul's Hospital, University of British Columbia

Bio: I completed my PhD at the Centre for High-Throughput Biology (CHiBi) at University of British Columbia (UBC), specializing in qualitative and quantitative proteomics. I am currently a postdoctoral fellow in the Department of Pathology and Laboratory Medicine at UBC working in the Clinical Chemistry Laboratory at St. Paul’s Hospital. My research focuses on translating biomarker research findings into quantitative mass spectrometric protein assays.

Authorship: Yu Zi (Emma) Zheng(1), Mari L. DeMarco(1,2)
(1) Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, Canada (2) Department of Pathology and Laboratory Medicine, St. Paul’s Hospital, Vancouver, Canada

Short Abstract

Protein digestion is a critical step in sample preparation prior to mass spectrometric analysis of proteins. Trypsin is the most commonly used protease in proteomics experiments; however digestion can be highly variable and is dependent on several factors including digestion buffer, denaturants, trypsin type, and sample type. Historically, trypsin digestion protocols have relied on lengthy digestion times, which are inappropriate for many clinical applications. We evaluated numerous iterations of digestion conditions for five plasma proteins and examined which changes yielded the greatest improvement in signal, reproducibility of the digestion profile, and rapid release of proteolytic peptides. It is our hope that this data can help clinical laboratorians accelerate the development phase of novel targeted assays by identifying practical approaches to improving digestion protocols.

Long Abstract


In current multiple reaction monitoring (MRM) protein assays, the biomarker of interest is often digested into peptides to facilitate mass spectrometry (MS) detection and measurement. This step is critical to assay performance as completeness, rate and stability of digestion can affect assay quality and turnaround time. While trypsin is the preferred protease due to its high cleavage specificity, digestion remains a variable process and a contributor to assay imprecision. Digestion is also often the rate-limiting step in quantification of proteins by MS. To help us better understand the key factors in the design of a quantitative protein MRM assay, we evaluated several variables that could affect the rate of peptide cleavage, digestion profile and/or signal intensity, including declustering potential (DP), collision energy (CE), trypsin type, digestion buffer, and denaturing conditions and additives.


We examined the tryptic digestion and MS detection of five proteins: apolioprotein A-1, retinol-binding protein 4, transthyretin, complement component 9 and C-reactive protein. In the design of our MRM method we used purified human proteins stocks and selected 2-9 unique tryptic peptides per protein, and monitored three transitions per peptide. We have translated the assay from purified protein stocks to plasma pools to evaluate the impact of modifications to the digestion procedure such as denaturing agent, temperature, enzyme:protein ratio, digestion buffers and trypsin types (sequencing-grade trypsin, bovine pancreatic trypsin or bovine pancreatic trypsin with chymotrypsin inhibitor TPCK). With consideration for assay turnaround time, we monitored digestion profiles by serially sampling digests for MRM analysis. To evaluate improvements in signal intensity, we calculated peak areas of all the transitions monitored and compared the values across different conditions/time points tested. Analysis was performed using a C18 analytical column operated at a flow rate of 0.25ml/min, and coupled to a triple quadrupole mass spectrometer (SCIEX 5500 QTRAP).


Using purified protein stocks, the detection sensitivity of our current MRM assay reaches the sub pmol to fmol range for the five protein targets. All transitions can be identified from less than 1 µl of plasma (~5-10 µg of total plasma protein), without the use of any off-line purification, enrichment or fractionation steps.

High temperature denaturation was found to be critical to generating a rapid reaction for the proteins studied. For plasma, ammonium bicarbonate was the preferred buffer over Tris buffer. Addition of glycerol did not significantly improve signal intensity but did improve sample stability if digests were stored at -80°C before analysis. TPCK trypsin produced signal intensity on par with sequencing grade trypsin. To generate rapid release of proteolytic peptides, TPCK trypsin outperformed sequencing-grade trypsin, giving a more rapid and stable profiles over the course of digestion; in some cases the digestion asymptote was reached 30 min to 2 h faster using TPCK versus sequencing-grade trypsin. Digestion profiles were monitored out to 24 hours, and with optimizations to the protocol the necessary digestion time was reduced to less than 20 min for all proteins of interest using a single digestion protocol. Several digestion profiles were observed including peptides that rapidly reached the digestion asymptote, peptides which decayed over the course of digestion, and peptides with anomalous but reproducible behavior (rising and falling in signal intensity several times over the course of the experiment).


We developed and optimized MRM methods for five diverse plasma/serum proteins. By iteratively testing variations to the digestion protocol and selecting rapidly generated and stable tryptic peptides, we were able to reduce the digestion time from 18 h to <20 min. As other researchers have shown different buffer preferences for serum, we are currently expanding the analysis to include serum. It is our hope that this data can help clinical laboratorians accelerate the development phase of novel targeted assays by identifying practical approaches to improving the rate and reproducibility of digestion profiles and peptide signal intensities.

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