Automation of nanoflow liquid chromatography‐tandem mass spectrometry for proteome analysis by using a strong cation exchange trap column

An approach was developed to automate sample introduction for nanoflow LC‐MS/MS (μLC‐MS/MS) analysis using a strong cation exchange (SCX) trap column. The system consisted of a 100 μm id×2 cm SCX trap column and a 75 μm id×12 cm C18 RP analytical column. During the sample loading step, the flow passing through the SCX trap column was directed to waste for loading a large volume of sample at high flow rate. Then the peptides bound on the SCX trap column were eluted onto the RP analytical column by a high salt buffer followed by RP chromatographic separation of the peptides at nanoliter flow rate. It was observed that higher performance of separation could be achieved with the system using SCX trap column than with the system using C18 trap column. The high proteomic coverage using this approach was demonstrated in the analysis of tryptic digest of BSA and yeast cell lysate. In addition, this system was also applied to two‐dimensional separation of tryptic digest of human hepatocellular carcinoma cell line SMMC‐7721 for large scale proteome analysis. This system was fully automated and required minimum changes on current μLC‐MS/MS system. This system represented a promising platform for routine proteome analysis.

[1]  S. Gygi,et al.  Automation of nanoscale microcapillary liquid chromatography-tandem mass spectrometry with a vented column. , 2002, Analytical chemistry.

[2]  J. Yates,et al.  Protein identification at the low femtomole level from silver-stained gels using a new fritless electrospray interface for liquid chromatography-microspray and nanospray mass spectrometry. , 1998, Analytical biochemistry.

[3]  T. Griffin,et al.  Evaluating preparative isoelectric focusing of complex peptide mixtures for tandem mass spectrometry-based proteomics: a case study in profiling chromatin-enriched subcellular fractions in Saccharomyces cerevisiae. , 2005, Analytical Chemistry.

[4]  J. Yates,et al.  An automated multidimensional protein identification technology for shotgun proteomics. , 2001, Analytical chemistry.

[5]  Richard D. Smith,et al.  Advanced nanoscale separations and mass spectrometry for sensitive high-throughput proteomics , 2005, Expert review of proteomics.

[6]  J. V. Van Beeumen,et al.  Automated nanoflow liquid chromatography/tandem mass spectrometric identification of proteins from Shewanella putrefaciens separated by two-dimensional polyacrylamide gel electrophoresis. , 2001, Rapid communications in mass spectrometry : RCM.

[7]  R. Aebersold,et al.  Mass spectrometry-based proteomics , 2003, Nature.

[8]  Hua Xiao,et al.  Electrochromatographic evaluation of a silica monolith capillary column for separation of basic pharmaceuticals , 2005, Electrophoresis.

[9]  H. Meiring,et al.  Nanoscale LC–MS(n): technical design and applications to peptide and protein analysis , 2002 .

[10]  E. Nägele,et al.  Two-dimensional nano-liquid chromatography-mass spectrometry system for applications in proteomics. , 2003, Journal of chromatography. A.

[11]  J. Yates,et al.  Large-scale analysis of the yeast proteome by multidimensional protein identification technology , 2001, Nature Biotechnology.

[12]  A. Shevchenko,et al.  Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. , 1996, Analytical chemistry.

[13]  Richard J. Simpson,et al.  Proteins and proteomics : a laboratory manual , 2003 .

[14]  Allis S. Chien,et al.  A double-vented tetraphasic continuous column approach to MuDPIT analysis on long capillary columns demonstrates superior proteomic coverage. , 2005, Journal of proteome research.

[15]  Ruedi Aebersold,et al.  Proteome analysis of low-abundance proteins using multidimensional chromatography and isotope-coded affinity tags. , 2002, Journal of proteome research.

[16]  S. Markey,et al.  Fully automated micro- and nanoscale one- or two-dimensional high-performance liquid chromatography system for liquid chromatography-mass spectrometry compatible with non-volatile salts for ion exchange chromatography. , 2005, Journal of chromatography. A.

[17]  M. Wilm,et al.  Electrospray and Taylor-Cone theory, Dole's beam of macromolecules at last? , 1994 .

[18]  Ruedi Aebersold,et al.  A microcapillary trap cartridge-microcapillary high-performance liquid chromatography electrospray ionization emitter device capable of peptide tandem mass spectrometry at the attomole level on an ion trap mass spectrometer with automated routine operation. , 2003, Rapid communications in mass spectrometry : RCM.

[19]  Patrick G. A. Pedrioli,et al.  A tool to visualize and evaluate data obtained by liquid chromatography-electrospray ionization-mass spectrometry. , 2004, Analytical chemistry.

[20]  C. Herberts,et al.  A microcapillary column switching HPLC-electrospray ionization MS system for the direct identification of peptides presented by major histocompatibility complex class I molecules. , 1998, Analytical chemistry.

[21]  John R Yates,et al.  Large-scale protein identification using mass spectrometry. , 2003, Biochimica et biophysica acta.

[22]  Richard D. Smith,et al.  High-efficiency nanoscale liquid chromatography coupled on-line with mass spectrometry using nanoelectrospray ionization for proteomics. , 2002, Analytical chemistry.

[23]  R. Aebersold,et al.  Mass spectrometry in proteomics. , 2001, Chemical reviews.

[24]  P. Thibault,et al.  Multiplex multidimensional nanoLC‐MS system for targeted proteomic analyses , 2005, Electrophoresis.

[25]  Hua Xiao,et al.  Preparation of monolithic silica column with strong cation-exchange stationary phase for capillary electrochromatography. , 2005, Journal of separation science.

[26]  J. Yates,et al.  Direct analysis of protein complexes using mass spectrometry , 1999, Nature Biotechnology.

[27]  K. Mechtler,et al.  Automated, on‐line two‐dimensional nano liquid chromatography tandem mass spectrometry for rapid analysis of complex protein digests , 2004, Proteomics.

[28]  S. Hanash,et al.  Multi-dimensional liquid phase based separations in proteomics. , 2003, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[29]  Joshua E. Elias,et al.  Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for large-scale protein analysis: the yeast proteome. , 2003, Journal of proteome research.

[30]  Xiaogang Jiang,et al.  Coupling the immobilized trypsin microreactor of monolithic capillary with muRPLC-MS/MS for shotgun proteome analysis. , 2006, Journal of proteome research.

[31]  F Gharahdaghi,et al.  Mass spectrometric identification of proteins from silver‐stained polyacrylamide gel: A method for the removal of silver ions to enhance sensitivity , 1999, Electrophoresis.