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p I ‐based phosphopeptide enrichment combined with nanoESI‐MS
Author(s) -
Hung ChienWen,
Kübler Dieter,
Lehmann Wolf D.
Publication year - 2007
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200600678
Subject(s) - phosphopeptide , chemistry , deamidation , chromatography , peptide , hydrolysis , trypsin , homogeneous , pi , enzyme , biochemistry , physics , thermodynamics
IEF is introduced as a new principle for enrichment and separation of phosphopeptides as obtained after digestion of phosphoproteins by trypsin. Tryptic peptides and phosphopeptides exhibit p I values, which overlap in the range of about 4–6. However, after methyl esterification of all carboxyl functions, the p I values of tryptic peptides and phosphopeptides regroup in discrete clusters. In addition, mono‐ and diphosphorylated peptides show different but very homogeneous p I values, with variations when internal Arg, Lys, or His residues are present. Experimentally, this new concept was applied for separation of model peptides on IPG strips pH 3–10 as used in the first dimension of 2‐DE. After IEF of methyl‐esterified peptides, the IPG strip was cut into pieces followed by peptide extraction, desalting and MS analysis by nanoESI‐MS. Phosphopeptides were found to focus in good agreement with their calculated p I values. This analytical strategy showed a resolution of about 0.2 p I units, and thus turned out to be capable of detecting minor differences in p I values, such as those occurring between pSer, pThr and pTyr residues. Using IPG strips with a p I range of 3–10, methyl esterified nonphosphorylated tryptic peptides are concentrated in the basic part of the IPG strip or even leave the strip. Thus, efficient enrichment of phosphopeptides and their subfractionation according to p I is obtained in one step. Minor hydrolytic side reactions including deamidation of Asn and partial hydrolysis of methyl esters are observed. The results show that IEF opens attractive avenues for the further advancement of analytical phosphoproteomics.