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Analytical and Bioanalytical Chemistry

, Volume 404, Issue 4, pp 1089–1101 | Cite as

Comparison of standard- and nano-flow liquid chromatography platforms for MRM-based quantitation of putative plasma biomarker proteins

  • Andrew J. Percy
  • Andrew G. Chambers
  • Juncong Yang
  • Dominik Domanski
  • Christoph H. BorchersEmail author
Original Paper

Abstract

The analytical performance of a standard-flow ultra-high-performance liquid chromatography (UHPLC) and a nano-flow high-performance liquid chromatography (HPLC) system, interfaced to the same state-of-the-art triple-quadrupole mass spectrometer, were compared for the multiple reaction monitoring (MRM)-mass spectrometry (MS)-based quantitation of a panel of 48 high-to-moderate-abundance cardiovascular disease-related plasma proteins. After optimization of the MRM transitions for sensitivity and testing for chemical interference, the optimum sensitivity, loading capacity, gradient, and retention-time reproducibilities were determined. We previously demonstrated the increased robustness of the standard-flow platform, but we expected that the standard-flow platform would have an overall lower sensitivity. This study was designed to determine if this decreased sensitivity could be compensated for by increased sample loading. Significantly fewer interferences with the MRM transitions were found for the standard-flow platform than for the nano-flow platform (2 out of 103 transitions compared with 42 out of 103 transitions, respectively), which demonstrates the importance of interference-testing when nano-flow systems are used. Using only interference-free transitions, 36 replicate LC/MRM-MS analyses resulted in equal signal reproducibilities between the two platforms (9.3 % coefficient of variation (CV) for 88 peptide targets), with superior retention-time precision for the standard-flow platform (0.13 vs. 6.1 % CV). Surprisingly, for 41 of the 81 proteotypic peptides in the final assay, the standard-flow platform was more sensitive while for 9 of 81 the nano-flow platform was more sensitive. For these 81 peptides, there was a good correlation between the two sets of results (R 2 = 0.98, slope = 0.97). Overall, the standard-flow platform had superior performance metrics for most peptides, and is a good choice if sufficient sample is available.

Figure

Optimization of sample loading on a standard- and b nano-flow systems; c comparison of plasma protein concentrations determined by both systems

Keywords

Nano-flow Standard-flow Plasma Stable isotope labeling Multiple reaction monitoring Quantitative proteomics 

Abbreviations

ACN

Acetonitrile

CE

Collision energy

CV

Coefficient of variation

CVD

Cardiovascular disease

CZE

Capillary zone electrophoresis

ELISA

Enzyme-linked immunosorbent assay

ESI

Electrospray ionization

FA

Formic acid

Fmoc

9-fluorenylmethoxycarbonyl

FWHM

Full width at half maximum

HPLC

High-performance liquid chromatography

LC

Liquid chromatography

LLOQ

Lower limit of quantitation

MALDI

Matrix-assisted laser desorption/ionization

MRM

Multiple reaction monitoring

MS

Mass spectrometry

MW

Molecular weight

NAT

Natural (endogenous)

Q1

First quadrupole

Q3

Third quadrupole

QqQ

Triple quadrupole

RP

Reversed-phase

R2

Coefficient of determination

RR

Relative response

SIS

Stable isotope-labeled standard

TOF

Time-of-flight

UHPLC

Ultra-high-performance liquid chromatography

XIC

Extracted ion chromatogram

Notes

Acknowledgments

The authors would like to thank Genome Canada, Genome BC, and the Western Economic Diversification of Canada for providing funding to the UVic Genome BC Proteomics Centre. The authors also recognize the fiscal, operational, and scientific support of the NCE CECR PROOF Centre of Excellence. We are grateful to Carol E. Parker for her assistance in preparing this manuscript. The authors declare no financial/commercial conflicts of interest.

Supplementary material

216_2012_6010_MOESM1_ESM.pdf (1.1 mb)
ESM 1 (PDF 1.09 MB)

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Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Andrew J. Percy
    • 1
  • Andrew G. Chambers
    • 1
  • Juncong Yang
    • 1
  • Dominik Domanski
    • 1
  • Christoph H. Borchers
    • 1
    • 2
    Email author
  1. 1.University of Victoria-Genome British Columbia Proteomics Centre, University of VictoriaVictoriaCanada
  2. 2.Department of Biochemistry and Microbiology, University of VictoriaVictoriaCanada

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