MSACL 2016 US Abstract

Strategies for Improving Specificity and Sensitivity for Multiplexed Proteomic Assays in for High-Flow, Fast-Gradient LC-MS in the Clinical Laboratory

Timothy Collier (Presenter)
Cleveland HeartLab Inc

Bio: Ph.D. - 2010, NC State University under David Muddiman in Quantitative Proteomics Post-Doc - 2010-2013, Washington University in St. Louis under Ron Bose using quantitative mass proteomics to characterize signal dynamics of drug resistant breast cancers. Currently Research and Development Scientist at Cleveland Heartlab under Cory Bystrom developing new quantitative proteomic assays of diagnostic or prognostic value in Cardiovascular Health.

Authorship: Timothy Collier, Cory Bystrom
Cleveland HeartLab, Cleveland, OH

Short Abstract

The potential for multiplexed proteomic assays is a key advantage of MS in the clinical laboratory. However, demands on throughput require rapid LC-MS, compressing analytical space and increasing chances for interference and suppression. Here, we describe the analytical challenges posed in the development of a high-flow, short-gradient, MRM method on a subset of the serum proteome, including the use of alternate enzymes and strategies to optimize sample preparation, the need to characterize analyte specific ion suppression, and contend with the possibility of nonspecific effects from co-eluting nominally isobaric species.

Long Abstract


A distinct advantage of mass spectrometry in the clinical laboratory is the potential for the development of multiplexed proteomic assays. Quantifying proteins and peptides in human serum and plasma is a challenging task given the complexity and large dynamic range needed for its characterization. Earlier work in the literature characterizing the specificity and sensitivity of MRM in the context of plasma proteomics demonstrate large dynamic range, and little non-specific crosstalk between target and non-specific analytes. However, most early studies have employed low-flow and long gradient chromatography, maximizing separation space to achieve those results. In a clinical laboratory environment, demands on throughput require LC-MS runs on a much shorter time-scale, compressing the analytical space and increasing the chances for interference and suppression. The work described here explores the analytical challenges posed in the development of a high-flow, short-gradient, MRM method on a subset of the serum proteome.


Human apolipoproteins were analyzed individually, in human serum, and in high density lipoprotein (HDL) purified from human serum. Proteins were digested with either trypsin or Lys-C. Transitions were optimized using Skyline. Liquid Chromatography was performed using an Agilent 1260 HPLC system equipped with a Phenomenex Kinetex C18 2.6 um 100 A column (2.6 mm i.d. x 50 mm) operating at a flow rate of 0.6 mL/min with a typical gradient spanning 5 to 50 % mobile phase B (0.1% formic acid in acetonitrile) in mobile phase A (0.1% formic acid in water) over 5 minutes. Detection was performed on an Agilent 6490 triple quadrupole mass spectrometer equipped with a jet stream electrospray source and operated in MRM or dynamic MRM mode with up to 114 transitions monitored per run. Peak Integration and quantification was performed using Agilent MassHunter Quantitative Analysis software and Skyline.


Trypsin is the most widely employed proteolytic enzyme for peptide mass spectrometry analysis. It is inexpensive and exhibits very specific cleavage behavior. We employed multiple grades of trypsin in the development of an assay targeting apolipoproteins from human serum. Our observations of tryptic products from individual purified apolipoproteins showed problematic abundances of missed cleavage products, impacting method sensitivity. Additionally, several target peptides did not reach a digestion endpoint after overnight digestion at a high enzyme-substrate ratio. We turned to Lys-C as an alternative digestion enzyme and achieved improved cleavage fidelity and favorable digestion kinetics, enabling our entire experimental workflow to be executed in hours rather than relying on overnight digestion.

We evaluated peptide target ion suppression as a result of sample matrix effects by post-column infusion of a peptide internal standard mixture. Each peptide monitored in the experiment showed varying regions of signal enhancement and suppression. Surprisingly, even in a less complex sample (enriched lipoprotein pools), several peaks appear from the eluting sample in the mass channels monitored for the internal standards. In a specific case, we observed the co-elution of a target and interfering peptide that differed in precursor mass by 0.3 m/z (1 Da) and shared several fragment ion masses. This is indicative of the confined m/z and chromatographic space of a typical clinical diagnostic workflow.


The use of MRM for the development of multiplexed proteomic assays holds enormous potential for application in high-throughput clinical laboratory environment, but requires increased experimental rigor to ensure specificity and sensitivity. This may require the use of alternative enzymes and conditions for sample preparation, full characterization of chromatographic and sample matrix effects on analyte detection, and careful selection of transitions to contend with the possibility of nonspecific effects from co-eluting nominally isobaric species.

References & Acknowledgements:

Financial Disclosure

SalaryyesCleveland HeartLab, Inc.
Board Memberno

IP Royalty: no

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