Rapid and sensitive separation of trace level protein digests using microfabricated devices coupled to a quadrupole-time-of-flight mass spectrometer

Citation
Jj. Li et al., Rapid and sensitive separation of trace level protein digests using microfabricated devices coupled to a quadrupole-time-of-flight mass spectrometer, ELECTROPHOR, 21(1), 2000, pp. 198-210
Citations number
40
Categorie Soggetti
Chemistry & Analysis
Journal title
ELECTROPHORESIS
ISSN journal
01730835 → ACNP
Volume
21
Issue
1
Year of publication
2000
Pages
198 - 210
Database
ISI
SICI code
0173-0835(200001)21:1<198:RASSOT>2.0.ZU;2-S
Abstract
The application of microfabricated devices coupled to a quadrupole time-of- flight mass spectrometer (Qq-TOF-MS) is presented for the analysis of trace level digests of gel-isolated proteins. In order to enhance the sample loa ding for proteomics analyses, two different on-chip sample preconcentration techniques were evaluated. First, a sample stacking procedure that used po larity switching to remove the sample buffer prior to zone electrophoresis was easily integrated on the microfabricated devices. With the present chip design, this preconcentration technique provided up to 70 nL sample inject ion with sub-nu detection limits for most peptide standards. For applicatio ns requiring larger sample loading, a disposable adsorption preconcentrator using a C-18 membrane is incorporated outside the chip. This preconcentrat ion method yielded lower peptide recoveries than that obtainable with sampl e stacking, and provided a convenient means of injecting several mu L Of Sa mple with detection limits of typically 2.5 nM for hydrophobic peptides. Th e analytical merits of both sample enrichment approaches are described for the identification of bands isolated from two-dimensional (2-D) gel separat ion of protein extracts from Haemophilus influenzae. Accurate molecular mas s measurements (< 5 ppm) in peptide mapping experiments is obtained by intr oducing an internal standard via a post-separation channel. Rapid identific ation of trace level peptides is also demonstrated using on-line tandem mas s spectrometry and database searching with peptide sequence tags*.