Enhanced Detection of Multiply Phosphorylated Peptides and Identification of Their Sites of Modification

Phosphorylation
is an important post-translational modification
that rapidly mediates many cellular events. A key to understanding
the dynamics of the phosphoproteome is localization of the modification
site(s), primarily determined using LC-MS/MS. A major technical challenge
to analysis is the formation of phosphopeptide–metal ion complexes
during LC which hampers phosphopeptide detection. We have devised
a strategy that enhances analysis of phosphopeptides, especially multiply
phosphorylated peptides. It involves treatment of the LC system with
EDTA and 2D-RP/RP-nanoUPLC-MS/MS (high pH/low pH) analysis. A standard
triphosphorylated peptide that could not be detected with 1D-RP-nanoUPLC-MS/MS,
even if the column was treated with EDTA-Na2 or if 25 mM
EDTA-Na2 was added to the sample, was detectable at less
than 100 fmol using EDTA-2D-RP/RP-nanoUPLC-MS/MS. Digests of α-casein and ß-casein were analyzed
by EDTA-1D-RP-nanoUPLC, 2D-RP/RP-nanoUPLC, and EDTA-2D-RP/RP-nanoUPLC
to compare their performance in phosphopeptide analysis. With the
first two approaches, no tri- and tetraphosphopeptides were identified
in either α- or ß-casein sample. With
the EDTA-2D-RP/RP approach, 13 mono-, 6 di-, and 3 triphosphopeptides
were identified in the α-casein sample, while
19 mono-, 8 di-, 4 tri-, and 3 tetraphosphopeptides were identified
in the ß-casein sample. Using EDTA-2D-RP/RP-nanoUPLC-MS/MS
to examine 500 μg of a human foreskin fibroblast cell lysate
a total of 1,944 unique phosphopeptides from 1,087 unique phosphoproteins
were identified, and 2,164 unique phosphorylation sites were confidently
localized (Ascore ≥20). Of these sites 79% were mono-, 20%
di-, and ∼1% were tri- and tetraphosphopeptides, and 78 novel
phosphorylation sites in human proteins were identified.