Assessing a novel microfluidic interface for shotgun proteome analyses.
Microfluidic interfaces coupled to ESI mass spectrometers hold great potential for proteomics as they have been shown to augment the overall sensitivity of measurements and require only a minimum of operator manipulations as compared to conventional nano-LC interfaces. Here, we evaluated a new type of HPLC-Chips holding larger enrichment columns (thus an increased sample loading capacity) for gel-free proteome studies. A tryptic digest of a human T-cell proteome was fractionated by strong cation exchange chromatography and selected fractions were analyzed by MS/MS on an IT mass spectrometer using both the new HPLC-Chip as well as a conventional nano-LC-MS/MS interface. Our results indicate that the HPLC-Chip is capable of handling very complex peptide mixtures and, in fact, leads to the identification of more peptides and proteins as compared to when a conventional interface was used. The HPLC-Chip preferentially produced doubly charged tryptic peptides. We further show that MS/MS spectra of doubly charged tryptic peptide ions are more readily identified by MASCOT as compared to those from triply charged precursors and thus argue that besides the improved chromatographic conditions provided by the HPLC-Chip, its peptide charging profile might be a secondary factor leading to an increased proteome coverage.
Staes
An
A
Department of Medical Protein Research, VIB, Ghent, Belgium.
Timmerman
Evy
E
Van Damme
Jozef
J
Helsens
Kenny
K
Vandekerckhove
Joël
J
Vollmer
Martin
M
Gevaert
Kris
K
D002458:Cell Fractionation; D006801:Humans; D019169:Jurkat Cells / Q000737:chemistry; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods; D008422:Materials Testing; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation; D010455:Peptides / Q000032:analysis; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods; D012984:Software
D010455:Peptides; D020543:Proteome