Document Detail


On-line amino acid-based capillary isoelectric focusing-ESI-MS/MS for protein digests analysis.
MedLine Citation:
PMID:  23062442     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Guijie Zhu; Liangliang Sun; Ping Yang; Norman J Dovichi
Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural     Date:  2012-04-27
Journal Detail:
Title:  Analytica chimica acta     Volume:  750     ISSN:  1873-4324     ISO Abbreviation:  Anal. Chim. Acta     Publication Date:  2012 Oct 
Date Detail:
Created Date:  2012-10-15     Completed Date:  2013-02-28     Revised Date:  2013-11-06    
Medline Journal Info:
Nlm Unique ID:  0370534     Medline TA:  Anal Chim Acta     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  207-11     Citation Subset:  IM    
Copyright Information:
Copyright © 2012 Elsevier B.V. All rights reserved.
Affiliation:
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA.
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MeSH Terms
Descriptor/Qualifier:
Amino Acids / 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:
R01 GM096767/GM/NIGMS NIH HHS; R01 RR031475/RR/NCRR NIH HHS; R21 GM103462/GM/NIGMS NIH HHS; R21 RR032362/RR/NCRR NIH HHS
Chemical
Reg. No./Substance:
0/Amino Acids; 0/Peptides; 0/Proteins; 0/Serum Albumin, Bovine; EC 3.4.21.4/Trypsin
Comments/Corrections

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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