| On-line amino acid-based capillary isoelectric focusing-ESI-MS/MS for protein digests analysis. | |
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MedLine Citation:
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PMID: 23062442 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Six amino acids with pIs that ranged from 3.2 to 9.7 were used as ampholytes to establish a pH gradient in capillary isoelectric focusing. This amino acid-based capillary isoelectric focusing (cIEF) was coupled with ESI-MS/MS using an electrokinetically pumped sheath-flow interface for peptide analysis. Amino acid-based isoelectric focusing generates a two-order of magnitude lower background signal than commercial ampholytes in the important m/z range of 300-1800. Good focusing was achieved for insulin receptor, which produced ~10 s peak width. For 0.1 mg mL(-1) bovine serum albumin (BSA) digests, 24±1 peptides (sequence coverage 47±4%) were identified in triplicate analysis. As expected, the BSA peptides were separated according to their pI. The concentration detection limit for the BSA digests is 7 nM and the mass detection limit is 7 fmole. A solution of six bovine protein tryptic digests spanning 5 orders of magnitude in concentration was analyzed by amino acid based cIEF-ESI-MS/MS. Five proteins with a concentration range spanning 4 orders of magnitude were identified in triplicate runs. Using amino acid based cIEF-ESI-MS/MS, 112 protein groups and 303 unique peptides were identified in triplicate runs of a RAW 264.7 cell homogenate protein digest. In comparison with ampholyte based cIEF-ESI-MS/MS, amino acid based cIEF-ESI-MS/MS produces higher resolution of five acidic peptides, much cleaner mass spectra, and higher protein spectral counts. |
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Authors:
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Guijie Zhu; Liangliang Sun; Ping Yang; Norman J Dovichi |
Publication Detail:
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Type: Journal Article; Research Support, N.I.H., Extramural Date: 2012-04-27 |
Journal Detail:
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Title: Analytica chimica acta Volume: 750 ISSN: 1873-4324 ISO Abbreviation: Anal. Chim. Acta Publication Date: 2012 Oct |
Date Detail:
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Created Date: 2012-10-15 Completed Date: 2013-02-28 Revised Date: 2013-04-16 |
Medline Journal Info:
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Nlm Unique ID: 0370534 Medline TA: Anal Chim Acta Country: Netherlands |
Other Details:
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Languages: eng Pagination: 207-11 Citation Subset: IM |
Copyright Information:
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Copyright © 2012 Elsevier B.V. All rights reserved. |
Affiliation:
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Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Amino Acids
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chemistry* Animals Cattle Cell Line Electrophoresis, Capillary Isoelectric Focusing Mice Peptides / analysis* Proteins / chemistry, metabolism* Serum Albumin, Bovine / chemistry, metabolism Spectrometry, Mass, Electrospray Ionization* Trypsin / metabolism |
| Grant Support | |
ID/Acronym/Agency:
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R01 RR031475/RR/NCRR NIH HHS; R21 GM103462/GM/NIGMS NIH HHS; R21 RR032362/RR/NCRR NIH HHS |
| Chemical | |
Reg. No./Substance:
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0/Amino Acids; 0/Peptides; 0/Proteins; 0/Serum Albumin, Bovine; EC 3.4.21.4/Trypsin |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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