Asp-Selective Microwave-Supported Acid Proteolysis - Proteomic and Metabolomic Approaches to Biomarker Discovery - page 223

A consistent aspect of the methods discussed
here is that they mainly take advantage of the
smaller peptides that are produced from Asp-
selective microwave-supported acid hydrolysis.
Although small peptides are easier to analyze
in existing high throughput work

identi ed in each fraction exhibit distinct

pro les. A control experiment without ltration

or charge selection identi ed 199 peptides.

Hauser and colleagues 15 demonstrated that
the longer peptides produced via Asp-selective
microwave-supported acid hydrolysis (12.5%
formic acid for 6 min at 130 C) can be activated
by electron-transfer dissociation (ETD) to
provide product ion spectra that can be used
to search databases. Electron transfer and elec-
tron capture fragmentation methods capitalize
on the electrostatic interactions between an elec-
tron and a positively charged peptide 30,31 and
demonstrate a high dependence on precursor
peptide charge state. Consequently, these are
more compatible with longer proteolytic prod-
ucts carrying more charges.

ows, as
Figure 2 indicates, one of the main bene

ts of
Asp-selective hydrolysis, or any single-residue-
speci c proteolytic method, is the production

of higher mass peptides. Longer peptides, with
molecular masses above 3,000 Da, require inno-
vative chromatography, higher resolution mass
analysis, enhanced ion activation, and novel
algorithms for spectral analysis. 18,25,26 -

cally, high resolution is required for electrospray
analysis of heavier peptides. This is due to the
higher charge states

Specifi-

4) observed in
precursor and product ion spectra and the use
of isotope spacing for automated charge assign-
ment and mass assignment. 12,27 In order to use
optimal conditions for both the heavier and the
lighter peptides in the product mixture of an
acid cleavage reaction, Cannon and Fenselau
have developed a work

(z

>

APPLICATIONS OF MICROWAVE-
SU PPORTED ACID HYDROLY SIS

Primary Sequence Studies of Human
Ribosomes
Because of the clear advantage offered by acid
cleavage for proteins with a high concentration of
Arg and Lys, Cannon and colleagues extended
the method to study the primary structures of
human ribosomes. 12 High-resolution analysis of
both precursor and fragment ions was employed
to accommodate the high mass peptides
produced in 12.5% acetic acid, 140

ow termed mass biased
partitioning in which peptides are bifurcated by
their size across a 3 kDa molecular weight cutoff

filter prior to injection for LC-MS/MS analysis. 28
The higher mass peptides are analyzed with
high resolution precursor and product ion
measurements, allowing charge state determina-
tion and accurate neutral mass determinations.
Data-dependent analysis parameters are con

g-
ured such that only analytes with charge states
of

5 C, 20
minutes. Of the 79 expected proteins, 70 were
con

þ 4 or greater are fragmented. The lower

ed from 366 distinct peptides,
in which 83% of peptide termini demonstrated
the expected cleavage. Most of the errant cleav-
ages observed occurred at Glu, Asn, and Gln resi-
dues. The role of deamidation in acid cleavage
has been further examined by Kaltashov and
colleagues. 32 Across all observed proteins, the
average sequence coverage was 46%.
N-terminal peptides were observed for 19 ribo-
somal proteins. Of those, 12 were acetylated
and 10 had lost the initiator methionine residue.

dently identi

mass flow-through sample is examined using

unit resolution. In our hands, high mass pep-
tides are most productively searched using
ProSightPC 29 and low mass peptides were
searched using PepArML ( https://edwardslab
.bmcb.georgetown.edu/pymsio/ ) . Analysis
of a whole-cell lysate from multiple myeloma
using acid cleavage and mass biased partitioning
resulted in the combined identi cation of 624

peptides from both fractions. Figure 3 con rms

that the peptide mass distributions of peptides