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Publication Details
AFRICAN RESEARCH NEXUS
SHINING A SPOTLIGHT ON AFRICAN RESEARCH
biochemistry, genetics and molecular biology
Off-line multidimensional liquid chromatography and auto sampling result in sample loss in LC/LC-MS/MS
Journal of Proteome Research, Volume 13, No. 8, Year 2014
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Description
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. © 2014 American Chemical Society.
Available Materials
https://efashare.b-cdn.net/share/pmc/articles/PMC4123945/bin/pr500530e_si_001.zip
https://efashare.b-cdn.net/share/pmc/articles/PMC4123945/bin/pr500530e_si_002.zip
https://efashare.b-cdn.net/share/pmc/articles/PMC4123945/bin/pr500530e_si_003.zip
Authors & Co-Authors
Magdeldin, Sameh
United States, San Diego
Scripps Research Institute
Japan, Niigata
Niigata University, Graduate School of Medical and Dental Science
Egypt, Ismailia
Faculty of Veterinary Medicine
Moresco, James J.
United States, San Diego
Scripps Research Institute
Yamamoto, Tadashi
Japan, Niigata
Niigata University, Graduate School of Medical and Dental Science
Yates, John R.R.
United States, San Diego
Scripps Research Institute
Statistics
Citations: 53
Authors: 4
Affiliations: 3
Identifiers
Doi:
10.1021/pr500530e
ISSN:
15353893
e-ISSN:
15353907
Research Areas
Health System And Policy