Quantitative Proteomics in Development of Disease Protein Biomarkers - Proteomic and Metabolomic Approaches to Biomarker Discovery - page 262

proteome digests; this approachprovides ameans
to normalize the differential loss of peptides from
preparation to preparation, increasing the quanti-
tation accuracy and precision of biomarker
proteins in a particular human sample.
However, the development of MRM quanti-
tation of large numbers of signature peptides
of protein biomarkers (biomarker candidates)
using AQUA peptides,

at varying degrees and reducing the quantita-
tion precision and accuracy.
Therefore, when possible, it is preferred to
add the protein reference standards early, such
as SILAC, super-SILAC, or SILAP proteins.
LC-SID-MRM-MS quantitation of signature
peptides can thus increase the accuracy and
precision, by using better protein reference
standards d stable isotope labeled full-length
proteins. SILAC proteomes have been used for
understanding disease mechanisms and discov-
ering new biomarkers in human samples. 72 e 76
In comparison to a SILAC proteome made of
a human cell type, super-SILAC and SILAP pro-
teomes provide the increased number of labeled
reference proteins for quantifying proteins in
complex human tissue and blood
samples. 77 e 79,85 Isotopically labeled, full-length
human proteins can also be made via bacteria/
yeast/cell-free expression. These types of tech-
nologies can produce labeled reference proteins
at reduced cost, although the labeled proteins
lack human-like post-translation modi

can be expensive.

MRM MS independently quanti es individual

peptides in a mixture, regardless of the labeling
status of the peptides. Therefore, chromato-
graphic separation of endogenous peptides
and their corresponding labeled reference
peptides is less a concern. Use of less-
expensive 2 H-labels reduces the cost of analysis,
although application of the 2 H-labeled reference
peptides in targeted quantitation of signature
peptides in complex digests of human samples
is yet to be demonstrated. 169 Isotopically
labeled reference peptides can also be made
by chemical derivatization of a control sample,
a less expensive option. Chemicals represented
by mass differential tags for relative and abso-
lute quanti

cations
and thus have limited application in quantifying
protein biomarkers based on changes in post-
translational modi

cation (mTRAQ) reagents have
been demonstrated for several MRM applica-
tions. 57,170,171 It is important to note, however,
that labeling peptides via chemical derivatiza-
tion brings in additional sources for differential
passage of quantitative information of the
original samples. It is yet to be seen if such
a strategy attracts broader interest for biomarker
development applications.
True absolute quantitation of protein
biomarkers in high complex human samples
like plasma requires normalizations in
protein-level sample preparation and protein
digestion. The precision in the digestion degree
for a particular protein can be larger than ideal,
e.g., more than 20% coef

cations. In addition, concate-
nation of signature peptides for different
proteins is also designed for preparation of
quanti cation concatamer (QconCAT) proteins

carrying stable isotope labels. Upon spiking the
QconCAT protein reference and digesting the
spiked proteome sample, reference signature
peptides for multiple biomarker proteins are
produced at equal moles. 174,175

High Resolution and High Mass
Accuracy MS
In the near future, new high-performance
mass spectrometers, 143,144,176 e 178 represented by
Orbitrap

cientofvariance
(CV), which makes the digestion variation the
bottleneck for accurate protein quantita-
tion. 127,172 This is particularly true for hard-
to-digest proteins and protein domains, 173
resulting in the signature peptide generation

and new generations of TOF mass
spectrometers, will play an increasing role in tar-
geted biomarker quantitation. The common
characteristic for targeted quantitation enabled
by the new generation of mass spectrometers is