Online Peptide fractionation using a multiphasic microfluidic liquid chromatography chip improves reproducibility and detection limits for quantitation in discovery and targeted proteomics

TY - JOUR

T1 - Online Peptide fractionation using a multiphasic microfluidic liquid chromatography chip improves reproducibility and detection limits for quantitation in discovery and targeted proteomics

AU - Krisp, Christoph

AU - Yang, Hao

AU - van Soest, Remco

AU - Molloy, Mark P

N1 - © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

PY - 2015/6

Y1 - 2015/6

N2 - Comprehensive proteomic profiling of biological specimens usually requires multidimensional chromatographic peptide fractionation prior to mass spectrometry. However, this approach can suffer from poor reproducibility because of the lack of standardization and automation of the entire workflow, thus compromising performance of quantitative proteomic investigations. To address these variables we developed an online peptide fractionation system comprising a multiphasic liquid chromatography (LC) chip that integrates reversed phase and strong cation exchange chromatography upstream of the mass spectrometer (MS). We showed superiority of this system for standardizing discovery and targeted proteomic workflows using cancer cell lysates and nondepleted human plasma. Five-step multiphase chip LC MS/MS acquisition showed clear advantages over analyses of unfractionated samples by identifying more peptides, consuming less sample and often improving the lower limits of quantitation, all in highly reproducible, automated, online configuration. We further showed that multiphase chip LC fractionation provided a facile means to detect many N- and C-terminal peptides (including acetylated N terminus) that are challenging to identify in complex tryptic peptide matrices because of less favorable ionization characteristics. Given as much as 95% of peptides were detected in only a single salt fraction from cell lysates we exploited this high reproducibility and coupled it with multiple reaction monitoring on a high-resolution MS instrument (MRM-HR). This approach increased target analyte peak area and improved lower limits of quantitation without negatively influencing variance or bias. Further, we showed a strategy to use multiphase LC chip fractionation LC-MS/MS for ion library generation to integrate with SWATH(TM) data-independent acquisition quantitative workflows. All MS data are available via ProteomeXchange with identifier PXD001464.

AB - Comprehensive proteomic profiling of biological specimens usually requires multidimensional chromatographic peptide fractionation prior to mass spectrometry. However, this approach can suffer from poor reproducibility because of the lack of standardization and automation of the entire workflow, thus compromising performance of quantitative proteomic investigations. To address these variables we developed an online peptide fractionation system comprising a multiphasic liquid chromatography (LC) chip that integrates reversed phase and strong cation exchange chromatography upstream of the mass spectrometer (MS). We showed superiority of this system for standardizing discovery and targeted proteomic workflows using cancer cell lysates and nondepleted human plasma. Five-step multiphase chip LC MS/MS acquisition showed clear advantages over analyses of unfractionated samples by identifying more peptides, consuming less sample and often improving the lower limits of quantitation, all in highly reproducible, automated, online configuration. We further showed that multiphase chip LC fractionation provided a facile means to detect many N- and C-terminal peptides (including acetylated N terminus) that are challenging to identify in complex tryptic peptide matrices because of less favorable ionization characteristics. Given as much as 95% of peptides were detected in only a single salt fraction from cell lysates we exploited this high reproducibility and coupled it with multiple reaction monitoring on a high-resolution MS instrument (MRM-HR). This approach increased target analyte peak area and improved lower limits of quantitation without negatively influencing variance or bias. Further, we showed a strategy to use multiphase LC chip fractionation LC-MS/MS for ion library generation to integrate with SWATH(TM) data-independent acquisition quantitative workflows. All MS data are available via ProteomeXchange with identifier PXD001464.

KW - Cell Line

KW - Cell Line, Tumor

KW - Chromatography, Liquid

KW - Humans

KW - Limit of Detection

KW - Microfluidics

KW - Peptides

KW - Proteomics

KW - Reproducibility of Results

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1074/mcp.M114.046425

DO - 10.1074/mcp.M114.046425

M3 - SCORING: Journal article

C2 - 25850434

VL - 14

SP - 1708

EP - 1719

JO - MOL CELL PROTEOMICS

JF - MOL CELL PROTEOMICS

SN - 1535-9476

IS - 6

ER -