Biomarker Discovery: Study Design and Execution - Proteomic and Metabolomic Approaches to Biomarker Discovery - page 10

involves protein precipitation and solubilization;
therefore, the downstream fractionation method
must be either electrophoresis or a liquid-phase
method.
Three different approaches for the global anal-
ysis of serum proteins have been used: global
serum proteome analysis using two- and
three-dimensional HPLC/MS 27,28 ; analysis of
low molecular weight proteins/peptides 29 ; and
investigation of proteins and peptides that are
bound to high-abundance serum proteins. 30
Unfortunately, studies have shown that the anal-
ysis of the plasma proteome by groups using
different methods resulted not only in different
number of protein identi

analysis. Each one of these steps can introduce
an error. The greatest error is introduced by the

final step, HPLC/MS/MS. It has been pointed
out 33,34 that to extract the largest number of
protein identi cations, the sample should be

analyzed at least in triplicate, because the
complexity of a digest of an entire proteome is
such that the analysis, evenwith a high-resolution
HPLC/MS system, exceeds the systems peak
capacity. 35 This observation was illustrated by
Dr. SamHanash and his coworkers in the analysis
of a plasma sample using HPLC/MS/MS. Repeat
runs resulted in the identi

cation of 32% and 36%
more peptides and proteins, respectively.

cations but poor over-
lap between the results. 31,32
Commonmethods for analysisof ametabolome
include GC/MS, HPLC/MS, or CE/MS. Which
technique to use depends on the compounds of
interest. Each technique has its advantages and
limitations. Buscher et al. 31 compared the three
techniques using a mixture of metabolites cover-
ing the pentose phosphate pathway, the tricarbox-
ylic acid cycle, redox metabolism, amino acids,
glycolysis, and nucleotides to test the three
methods. Out of 75 intermediate standard metab-
olites, 33 were common to the 3 methods, 64 by
CE, 42 by GC, and 65 by LC. A combination of
LC and GC detected 70 metabolites. All metabo-
lites were detected using the three methods. These
results prove that the method of analysis is an
important part of biomarker discovery.

Effect of Mass Spectrometer Type
on the Results
In proteomic and metabolomic studies, the
mass spectrometer plays a central role and the
selection of the instrument can affect the results.
Gika et al. 35 coupled a single ultra-high-pressure
liquid chromatography (UPLC) instrument to
a triple quadrupole linear ion trap (Q-TRAP) and
a hybrid quadrupole time-of- ight (Qq-TOF)

mass spectrometer using both positive and nega-
tive electrospray ionization (ESI) to study the
metabolic pro flow from the

UPLC column was split equally and the eluent
streams were simultaneously directed to the inlets
of the two mass spectrometers. Data from both
mass spectrometerswere subjected tomultivariate
statistical analysis. After applying the same data
extraction software, a number of ions were found
to be unique to either data set.
The study clearly indicates that not all ions
were detected by both the Qq-TOF and Q-TRAP
mass spectrometers. The authors concluded that
“

le of rat urine. The

Number of Replicates
The conventional teaching in analytical chem-
istry is that each sample should be analyzed in
triplicate and the mean and standard deviation
should be reported. Unfortunately, most pub-
lished proteomic and metabolomic studies
analyze each sample only once, which does not
permit the error in the measurement to be calcu-
lated. Proteomic analysis of a biological sample
involves depletion of high molecular weight
proteins, digestion, fractionation, and HPLC/MS

given the design differences between instru-
ments this is perhaps not that surprising a finding

but nevertheless it raises important questions
about how to evaluate data from different labora-
tories produced on different mass spectrometers
even when (nominally) the same sample process-
ing and chromatography have been used.

36

”