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.

Development of glutaraldehyde-crosslinked chymotrypsin and an in situ immobilized enzyme microreactor with peptide mapping by capillary electrophoresis.

Immobilized proteolytic enzymes present several advantages over their soluble form, not the least of which is suppression of autoproteolysis peaks even at high enzyme-to-substrate ratios. We have made immobilized chymotrypsin by directly crosslinking it with glutaraldehyde to produce polymeric particles. Digestion of two model substrates using the particles was followed by CE peptide mapping with detection by UV absorbance or LIF. Results showed that autoproteolysis was highly suppressed and that different storage conditions of the particles in the short term (24 h) did not affect digestion of denatured BSA. As well, the chymotrypsin particles were indifferent to the presence of fluorescein groups on a casein substrate. Glutaraldehyde crosslinking of chymotrypsin inside a fused silica capillary column to make an immobilized enzyme reactor (IMER) was achieved in a series of reagent addition and washing steps, entirely automated using a commercial CE instrument. Digestion of myoglobin in the IMER for 30 min at 37°C followed by peptide mapping by CE-MS of the collected digest allowed identification of 17 chymotryptic peptides of myoglobin, or 83% primary sequence coverage.