Off-Line Multidimensional Liquid Chromatography and Auto Sampling Result in Sample Loss in LC/LC–MS/MS

Large-scale
proteomics often employs two orthogonal separation
methods to fractionate complex peptide mixtures. Fractionation can
involve ion exchange separation coupled to reversed-phase separation
or, more recently, two reversed-phase separations performed at different
pH values. When multidimensional separations are combined with tandem
mass spectrometry for protein identification, the strategy is often
referred to as multidimensional protein identification technology
(MudPIT). MudPIT has been used in either an automated (online) or
manual (offline) format. In this study, we evaluated the performance
of different MudPIT strategies by both label-free and tandem mass
tag (TMT) isobaric tagging. Our findings revealed that online MudPIT
provided more peptide/protein identifications and higher sequence
coverage than offline platforms. When employing an off-line fractionation
method with direct loading of samples onto the column from an eppendorf
tube via a high-pressure device, a 5.3% loss in protein identifications
is observed. When off-line fractionated samples are loaded via an
autosampler, a 44.5% loss in protein identifications is observed compared
with direct loading of samples onto a triphasic capillary column.
Moreover, peptide recovery was significantly lower after offline fractionation
than in online fractionation. Signal-to-noise (S/N) ratio, however,
was not significantly altered between experimental groups. It is likely
that offline sample collection results in stochastic peptide loss
due to noncovalent adsorption to solid surfaces. Therefore, the use
of the offline approaches should be considered carefully when processing
minute quantities of valuable samples.