Sample simplification through affinity selection in proteomics

2004 2004

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Abstract (summary)

In “bottom up” approach in proteomics, it is always challenging to analyze the large number of peptides generated by protein digestion. Affinity selection of specific peptides provides a quick way to reduce sample complexity to match the analytical capacity of the system. In this thesis, agarose based immobilized metal affinity chromatography (IMAC) columns loaded with copper (II) were evaluated for the selection of histidine-containing peptides in comparative proteomics. Recovery, binding specificity, and reproducibility were investigated with the model proteins, apo-transferrin and β-galactosidase. The study showed that Cu(II)-IMAC is a robust sample simplification method with little non specific binding and reasonable column longevity. In addition, it was found out that the selectivity could be tuned by the eluting condition, salt concentration, peptide N-terminus acylation, and type of sorbent. As an application, Cu(II)-IMAC in tandem with reversed phase chromatography RPC) was applied to enrich histidine-rich peptides and to quantify a yeast protein extract at two fermentation times. An inverse labeling strategy was applied to increase reliability in determining upand down-regulation.

Two other methods were investigated as well. One was strong anion exchange chromatography coupled with Cu(II)-IMAC. Another was strong cation exchange of cysteine derivatized peptides. Due to the fact that most tryptic peptides carry a negative charge at neutral pH, cysteine residues were derivatized with a quaternary amine tag (QAT) and selected by strong cation exchange chromatography. An advantage of the method is that it enriched QAT derivatized cysteinyl peptides. Compared with other cysteine selection methods, this approach provides a simple, fast and robust way to facilitate enrichment cysteinyl peptide.

Indexing (details)

Analytical chemistry
0486: Analytical chemistry
Identifier / keyword
Pure sciences; Cysteinyl peptide; Immobilized metal affinity chromatography; Proteomics
Sample simplification through affinity selection in proteomics
Ren, Diya
Number of pages
Publication year
Degree date
School code
DAI-B 65/10, Dissertation Abstracts International
Place of publication
Ann Arbor
Country of publication
United States
0496111450, 9780496111459
Regnier, Fred E.
Purdue University
University location
United States -- Indiana
Source type
Dissertations & Theses
Document type
Dissertation/thesis number
ProQuest document ID
Database copyright ProQuest LLC; ProQuest does not claim copyright in the individual underlying works.
Document URL
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