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
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*.