Improved particle-packed HPLC/MS microchips for proteomic analysis.
The influence of packing process parameters (packing pressure, application of ultrasound) and the stationary phase particle size (3.5 and 5 μm) on the chromatographic performance of HPLC/MS chips was systematically investigated for proteomic samples. First, reproducibility and detection limits of the separation were evaluated with a low-complexity sample of tryptic BSA peptides. The influence of adsorbent packing quality on protein identification was then tested with a typical proteomics sample of high complexity, a human plasma protein fraction (Cohn fraction IV-4). All HPLC/MS chips provided highly reproducible separations of these proteomic samples, but improved packing conditions and smaller particle sizes resulted in chromatograms with narrower peaks and correspondingly higher signal intensities. Improved separation performance increased the peak capacity, the number of identified peptides, and thus the sequence coverage in the proteomic samples, particularly for low sample amounts.
Department of Clinical Chemistry, University Medical Center Hamburg - Eppendorf, Hamburg, Germany.
Maria
M
Trusch
Steffen
S
Ehlert
Andreas
A
Bertsch
Oliver
O
Kohlbacher
Diana
D
Hildebrand
Hartmut
H
Schlüter
Ulrich
U
Tallarek
D001798:Blood Proteins / Q000737:chemistry / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods; D006801:Humans; D056656:Lab-On-A-Chip Devices; D046208:Microchip Analytical Procedures / Q000379:methods; D040901:Proteomics / Q000295:instrumentation / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods; D014357:Trypsin / Q000378:metabolism
D001798:Blood Proteins; C032309:Cohn fraction IV; D014357:Trypsin