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


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.


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


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





Collision energy


Coefficient of variation


Cardiovascular disease


Capillary zone electrophoresis


Enzyme-linked immunosorbent assay


Electrospray ionization


Formic acid




Full width at half maximum


High-performance liquid chromatography


Liquid chromatography


Lower limit of quantitation


Matrix-assisted laser desorption/ionization


Multiple reaction monitoring


Mass spectrometry


Molecular weight


Natural (endogenous)


First quadrupole


Third quadrupole


Triple quadrupole




Coefficient of determination


Relative response


Stable isotope-labeled standard




Ultra-high-performance liquid chromatography


Extracted ion chromatogram



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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