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.
Conventional capillary liquid chromatography/mass spectrometry (LC/MS) typically employs low microl/min flow rates with gas/liquid sheath to enhance spray stability. Over the past several years a number of reports have demonstrated success with electrospray (ES) interface designs optimized for submicroliter/min flows which have clear advantages in terms of enhancement of detection limit, lower sample consumption, and ability to accommodate a wider range of buffer conditions. We report here a fritless electrospray interface (FESI) design that is inexpensive and robust and can be operated and adapted to accommodate a variety of applications for submicroliter/min flow rates. The novelty of this interface revolves around the use of a fritless microcapillary column and precolumn application of electrospray voltage at a microtee junction to achieve stable microspray and nanospray flow rates. This sheathless FESI device eliminates postcolumn dead volume since small particles (</= 10 micron) are packed directly into laser-pulled fused silica capillary needles from which a spray originates. For analysis of proteins/peptides in solution, low femtomole sensitivity has been achieved (attomoles for selected-ion monitoring), while low nanogram sensitivity was attained for proteins derived from in-gel-digested silver-stained bands from 1-D and 2-D gels. Several applications for tandem MS protein/peptide identification using LC-microspray, LC-nanospray, or infusion nanospray are presented.
Gatlin
C L
CL
Department of Molecular Biotechnology, University of Washington, Seattle, Washington 98195-7730, USA.
Kleemann
G R
GR
Hays
L G
LG
Link
A J
AJ
Yates
J R
JR
D000595:Amino Acid Sequence; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000032:analysis / Q000737:chemistry; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis / Q000737:chemistry; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D012680:Sensitivity and Specificity; D016622:Silver Staining / Q000379:methods*
D005656:Fungal Proteins; D010455:Peptides; D011506:Proteins