A microscale electrospray interface for on-line, capillary liquid chromatography/tandem mass spectrometry of complex peptide mixtures.
A microcapillary liquid chromatography (HPLC) system designed for the gradient elution of peptide and protein samples at flow rates < 1 microL/min has been coupled to a triple-sector quadrupole mass spectrometer via a simple sheathless electrospray interface (microspray). The microspray interface used a flame-drawn, uncoated, fused silica needle with tip outer diameters in the range of 15-20 microm and an opening less than 5 microm in diameter. Online sample filtration to prevent clogging of the drawn needle was accomplished by using a hydrophilic PVDF membrane filter integrated into the needle assembly. The spray potential (0.5-1 kV) was applied directly to the sample stream through the capillary union. Stable electrospray conditions were obtained over the full range of the gradient (0-90% acetonitrile in water) and was generally independent of flow rate. Both off-line and online analyses of proteins and peptide digest mixtures were performed at sample levels less than 10 fmol. HPLC parameters could be optimized for either rapid LC/MS analysis or enhanced performance in LC/MS/MS experiments by modulation of the eluting peak widths. Additionally, flow could be greatly reduced as selected components pass through the interface to prolong the time available to collect mass spectral data. The reduced spectral background and peak width manipulation facilitated the acquisition of peptide production spectra (MS/MS) using real-time, automated instrument control procedures.
Davis
M T
MT
Beckman Research Institute, City of Hope, Duarte, California 91010, USA.
Stahl
D C
DC
Hefta
S A
SA
Lee
T D
TD
D002851:Chromatography, High Pressure Liquid; D013058:Mass Spectrometry; D008567:Membranes, Artificial; D009862:Online Systems; D010455:Peptides / Q000032:analysis / Q000302:isolation & purification*; D011145:Polyvinyls / Q000737:chemistry; D015203:Reproducibility of Results; D005465:Fluorocarbon Polymers
D008567:Membranes, Artificial; D010455:Peptides; D011145:Polyvinyls; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers