MSACL 2019 EU Abstract

The Combined Use of Lys-C and Trypsin Provides Better Digestion Efficiency for MS-based Protein Quantitation

Fred P.H.T.M. Romijn (1), Maarten P.J. v. Hoorn (1), Zsuzsanna Kuklenyik (2), Julia Dittrich (3), Uta Ceglarek (3), L. Renee Ruhaak (1), Christa M. Cobbaert (1), on behalf of IFCC WG APO-MS
(1) Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center, Leiden, The Netherlands. (2) Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA, USA. (3) Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Germany.

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Fred Romijn (Presenter) Leiden University Medical Center

Relevant Financial Disclosures (within past 24 months) No relevant financial relationship(s) to disclose. Discussing Products? NO, presenter will NOT present educational content related to products/services of the ineligible company(ies) listed above. Conflict mitigation NOT REQUIRED.

INTRODUCTION: There is growing evidence that serum levels of apolipoproteins (apos) refines cardiovascular disease (CVD) risk assessment as compared to traditional blood-based lipid markers. Several research groups developed LC-MS strategies for quantitation of apos. An IFCC WG was recently established to set up a Reference Measurement System for 7 apolipoproteins. To this end the WG decided to develop a Reference Measurement Procedure using peptide-based primary Reference Materials and bottom up proteomics strategy. This strategy requires equimolar digestion of proteins to peptides to ensure metrological traceability, which is particularly challenging for apos C-I and C-III. The combined use of Lys-C and trypsin improves digestion for protein identification, but has not been evaluated for protein quantitation yet. Here we present the use of Lys-C in combination with trypsin to achieve equimolar digestion of apolipoproteins for bottom up proteomics quantification.
METHODS: A multiplex LC-MRM-MS method was developed, containing 29 peptides from apos A-I, (a), B, C-I, C-II, C-III and E. Stable isotope labelled peptides were added to serum samples (0.4 µL) and apos in human serum were denatured and reduced in the presence of 0.33% (w/v) DOC and 23 mM TCEP. Upon alkylation with 4.6 mM IAA, serum proteins were digested with 0.8 µg Sequencing Grade Modified Trypsin for 3 h at 37°C. Peptides were enriched using Oasis HLB solid phase extraction prior to LC-MRM-MS analysis. For digestion optimization, 0.28-0.02 µg of Mass Spec Grade Lys-C were added 2, 1 or 0.5 h prior to the initiation of trypsin digestion. Moreover, the amount of trypsin was optimized. Relative responses (RR) were calculated at digestion times of 0, 1, 3 and 21 h.
RESULTS: Compared to trypsin-only digestion, addition of Lys-C 1 h prior of trypsin is advantageous and results in faster digestion and higher peptide yields. Digestion of 0.4 µL serum with 0.12 µg Lys-C with 0.6 µg trypsin resulted in stable plateaus for all peptides after 3 h digestion time. Compared to trypsin-only digestion, peptide yields increased 100% for peptide EFGNT (apo C-I) and 55% for TPDVS (apo C-I), while increases 85% for GWVTD (apo C-III) and 45% for DYWST (apo C-III). Peptides from apo B(total) increased 0-50%, apo B100 peptides 0-20%, apo(a) peptides 10-50% and apo E peptides 0-10%. No significant differences in digestion were observed for peptides from apos A-I and C-II.
CONCLUSION: Lys-C (0.12 µg) added 1 hour prior to trypsin (0.6 µg) improved digestion efficiency in 5 out of 7 of apos in serum (0.4 µL) compared to trypsin-only digestion. This digestion protocol results in stable plateaus for apos A-I, (a), B, C-I, C-II, C-III and E after 3 h of trypsin digestion. The combined use of Lys-C and trypsin likely enables a common sample preparation protocol allowing multiplexed, MS-based standardization of 7 apos, although equimolarity of the digestion step still needs to be proven quantitatively.