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United States Department of Agriculture

Agricultural Research Service

Research Project: GENOMIC APPROACHES TO IMPROVING TRANSPORT AND DETOXIFICATION OF SELECTED MINERAL ELEMENTS IN CROP PLANTS Title: A Comparison of nLC-ESI-MS/MS and nLC-MALDI-MS/MS for GeLC-based Protein Identification and iTRAQ-based Shotgun Quantitative Proteomics

Authors
item Yang, Yong
item Howe, Kevin
item Zhang, Sheng - CORNELL UNIVERSITY
item Wilson, David - CORNELL UNIVERSITY
item Moser, Felix - CORNELL UNIVERSITY
item Irwin, Diana - CORNELL UNIVERSITY
item Thannhauser, Theodore

Submitted to: Journal of Biomolecular Techniques
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 11, 2007
Publication Date: September 14, 2007
Citation: Yang, Y., Howe, K.J., Zhang, S., Wilson, D., Moser, F., Irwin, D., Thannhauser, T.W. 2007. A comparison of nlc-esi-ms/ms and nlc-maldi-ms/ms for gelc-based protein identification and itraq-based shotgun quantitative proteomics. Journal of Biomolecular Techniques. 18:226-237.

Interpretive Summary: In this work we compare and contrast the performance of electrospray and matrix assisted laser desorption as an ionization interface for nano scale liquid chromatography-mass spectrometry analysis in two common proteomics applications: the analysis of one dimensional gel bands, so called "1-D GeLC" and the analysis of isotope coded strong cation exchange fractions, so called "quantitative shotgun proteomics." We discover that the two approaches are complementary and that in complex samples proteomic coverage increases as much as 45% by employing both over what can be achieved by using either one alone. Furthermore, the insensitivity of matrix assisted laser desorption ionization to ionization suppression agents was found to be a significant advantage because it can be used to detect the presence of such agents and reduce their deleterious effects.

Technical Abstract: The use of nLC-ESI-MS/MS in shotgun proteomics experiments and GeLC-MS/MS analysis is well accepted and routinely available in most proteomics laboratories. However, the same cannot be said for nLC-MALDI MS/MS, which has yet to experience such widespread acceptance despite the fact that the MALDI technology offers several critical advantages over ESI. As an illustration, in an analysis of moderately complex sample of E. coli proteins, the use MALDI in addition to ESI in GeLC-MS/MS resulted in a 16% average increase in protein identifications, while with more complex samples the number of additional protein identifications increased by an average of 45%. The size of the unique peptides identified by MALDI was, on average, 25% larger than the unique peptides identified by ESI and they were found to be slightly more hydrophilic. The insensitivity of MALDI to the presence of ionization suppression agents was shown to be a significant advantage, suggesting it be used as a complement to ESI when ion suppression is a possibility. Furthermore, the higher resolution of the TOF/TOF instrument improved the sensitivity, accuracy and precision of the data over that obtained using only ESI-based iTRAQ experiments using a linear ion trap. Nevertheless, accurate data can be generated with either instrument. These results demonstrate that coupling nanoLC with both ESI and MALDI ionization interfaces improves proteome coverage, reduces the deleterious effects of ionization suppression agents and improves quantitation, particularly in complex samples.

Last Modified: 12/18/2014
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