Asp-Selective Microwave-Supported Acid Proteolysis - Proteomic and Metabolomic Approaches to Biomarker Discovery - page 225

FIGURE 4 Partial MALDI-TOF spectrum demonstrating an approximately twofold increase in abundance of an isotope-
labeled adenovirus peptide produced by microwave-supported Asp-selective acid hydrolysis in 18 O water. Relative abun-
dances of each peak are provided in parentheses. (Reprinted with permission from reference #13.)

ow. 17 Asp-
selective acid digestion cleaves the SUMO modi-

acid hydrolysis into their work membrane proteins ed when Asp-

selective acid hydrolysis followed cyanogen
bromide cleavage, both in formic acid. 22
In a

identi

fier at most 11 residues (depending on which
SUMO protein) from the C terminus. The combi-
nation of trypsin and microwave-supported acid
hydrolysis together produced a target peptide in
which both branches could be characterized.
Successful analyses were obtained of di-SUMO-
1, of di-SUMO-2, and of SUMO-3 conjugated to
the protein E2 25K when the SUMO conjugates
were digested with 12.5% acetic acid at 140 C
for 10 minutes and then with trypsin. Successful
analysis was accomplished by both MALDI
tandem time-of-

final illustration of the value of selective
cleavage at Asp residues, acid digestion has
been incorporated by Alam and colleagues into
a method for rapid identi

cation of hemoglobin
variants from clinical samples. 10 The variants of
interest potentially contain new Lys or Arg resi-
dues, which create new tryptic cleavage sites and
complicate data analysis of tryptic biomarkers.
Asp-selective digestion provides cleavage sites
consistent between variants, and peptides that
differ only by changes in mass due to amino
acid substitution(s). Enzymatic digestion with
the metalloprotease AspN from Psuedomonas
fragi provides the necessary speci

ight (TOF) MS and electrospray
ionization (ESI) LC-MS/MS. Post-source decay,
CID, and ETD all provided suf

cient fragmenta-
tion. A tandem mass spectrum obtained using
CID is shown in Figure 5 , in which manual inter-
pretation identi

city but is
chemically incompatible with the collection of
blood samples in the presence of the anti-
coagulant ethylenediaminetetraacetic acid
(EDTA). AspN, requires a Zn þ 2 cofactor and is
inhibited by EDTA. 34 Asp-selective microwave-
supported acid hydrolysis (0.05 e 3.0% formic
acid, 150 C, 2 e 10 minutes) provides the same
speci

es fragment ions derived from
both sides of the branched peptide and locates
the site of SUMOylation.
High-throughput proteomic work ows have

also bene tted from combining microwave-

supported Asp-selective acid hydrolysis with
other proteolytic methods. Wu and colleagues
found a signi

city and the added bonus of short diges-
tion times. The resulting peptides were analyzed cant increase in the number of