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.

Reversed-phase microcapillary chromatography (RP-microLC) combined with electrospray ionization tandem mass spectrometry (ESI-MS/MS) is one of two prevailing techniques in proteomic analysis, the other being matrix-assisted laser desorption/ionization (MALDI). Despite the arguably better dynamic range obtainable with ESI, MALDI is increasingly popular due to ease of use, ruggedness and the ability to decouple separation from ionization. By contrast, in order to take advantage of the sensitivity and dynamic range afforded by the concentration-dependent nature of ESI, it is directly coupled to separations that take place in small i.d. RP-microLC columns. This gain in sensitivity often comes at a loss of ruggedness due to clogging of the small i.d. RP-microLC columns, one result of which is limited sample throughput. Here we describe a combined micropre-column-microLC-ESI device that is sensitive, rugged and modular in design allowing facile construction and troubleshooting. Due to low signal-to-noise as little as 1 attomole of a peptide can be selected by data-dependent methods for collision-induced dissociation. Importantly, the resulting tandem mass spectrum is of high enough quality to identify the peptide sequence by a database search against a complex database using SEQUEST. Finally, the device is demonstrated to be rugged as judged by >60 consecutive reversed-phase microLC separations on complex peptide mixtures before chromatographic resolution is degraded.

[1]  P. Matsudaira,et al.  Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. , 1987, The Journal of biological chemistry.

[2]  Richard D. Smith,et al.  Attomole level capillary electrophoresis-mass spectrometric protein analysis using 5 .mu.m i.d. capillaries , 1992 .

[3]  S. Gygi,et al.  An integrated microfluidics-tandem mass spectrometry system for automated protein analysis. , 1998, Analytical chemistry.

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

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

[6]  Richard D. Smith,et al.  Use of small‐diameter capillaries for increasing peptide and protein detection sensitivity in capillary electrophoresis‐mass spectrometry , 1993, Electrophoresis.

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

[8]  L. Deterding,et al.  Nanoscale packed-capillary liquid chromatography coupled with mass spectrometry using a coaxial continuous-flow fast atom bombardment interface. , 1991, Analytical chemistry.

[9]  Richard D. Smith,et al.  Reduced elution speed detection for capillary electrophoresis/mass spectrometry , 1993 .

[10]  J. Ramsey,et al.  Subattomole-Sensitivity Microchip Nanoelectrospray Source with Time-of-Flight Mass Spectrometry Detection. , 1999, Analytical chemistry.

[11]  M. Wilm,et al.  Analytical properties of the nanoelectrospray ion source. , 1996, Analytical chemistry.

[12]  Richard D. Smith,et al.  Sheathless capillary electrophoresis-electrospray ionization mass spectrometry using 10 mu m I.D. capillaries: analyses of tryptic digests of cytochrome c. , 1994, Journal of chromatography. A.

[13]  J. Jorgenson,et al.  Preparation and evaluation of packed capillary liquid chromatography columns with inner diameters from 20 to 50 μm , 1989 .

[14]  R. Caprioli,et al.  Micro-electrospray mass spectrometry: Ultra-high-sensitivity analysis of peptides and proteins , 1994, Journal of the American Society for Mass Spectrometry.

[15]  J. Yates,et al.  Direct analysis and identification of proteins in mixtures by LC/MS/MS and database searching at the low-femtomole level. , 1997, Analytical chemistry.

[16]  Ruedi Aebersold,et al.  Quantitative proteomic analysis of Myc oncoprotein function , 2002, The EMBO journal.

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

[18]  Terry D. Lee,et al.  Rapid protein identification using a microscale electrospray LC/MS system on an ion trap mass spectrometer , 1998, Journal of the American Society for Mass Spectrometry.

[19]  R. Aebersold,et al.  Approaching complete peroxisome characterization by gas‐phase fractionation , 2002, Electrophoresis.

[20]  J R Yates,et al.  Protein sequencing by tandem mass spectrometry. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[21]  J. Shabanowitz,et al.  Subfemtomole MS and MS/MS peptide sequence analysis using nano-HPLC micro-ESI fourier transform ion cyclotron resonance mass spectrometry. , 2000, Analytical chemistry.

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

[23]  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.