Biomarker Discovery: Study Design and Execution - Proteomic and Metabolomic Approaches to Biomarker Discovery - page 11

In another study, Elias et al. 33 compared the
results of triplicate measurements of the yeast
proteome by LC-MS/MS using linear ion
trap (LTQ) and Qq-TOF mass spectrometers.
The data was searched using both Mascot and
SEQUEST. The results from the two instruments
were different, with each search engine providing
a different number of identi

resolving power, two-dimensional 2D-HPLC is
the preferred procedure for the comprehensive
separation of the proteome. 37,38

Errors in Measurements
One cannot ignore the experimental and
human errors in the measurement of proteins
and metabolites in complex mixtures. In a recent
metabolic study usingGC/MS to search for amino
acidmarkers in urine of 11 bladder cancer patients
and 8 controls, the error of reported results was
extremely high and ranged from 4% to 93% and
6% to 94% for the patients with bladder cancer
and controls, respectively. 39 The high errors and
the overlap between cancer patients and controls
do not result in a speci

cations. From the

LTQ data, 666 and 644 identi cations were exclu-

sive to Mascot and SEQUEST, respectively, and
4,056 proteins were identi ed using both algo-

rithms. For the Qq-TOF data, 1,012 and 510 iden-
ti

cations were exclusive to Mascot and
SEQUEST, respectively, and 1,955 proteins were
identi ed using both algorithms. 34

c and sensitive method,
nor can they be used for population studies or to
replace a clinical test. Therefore, attention should
be paid to eliminate human and experimental
errors. Errors arise from sample collection and
preparation procedures and analysis.

Effect of Separation Instrumentation
on the Results
The most commonly used analytical methods
for finding potential biomarkers are SDS-PAGE,

HPLC/MS, and GC/MS. SDS-PAGE is used
only for the fractionation and separation of
proteins. GC is an excellent technique for the
separation of volatile compounds; however, it is
not suitable for the separation of proteins. It is
a simple, relatively economical, and fast tech-
nique that possesses high resolving power and
reproducibility. Although GC using a single
column can achieve high resolution separations,
2D-GC is the preferred procedure for the compre-
hensive separation of metabolomic mixtures. 37
HPLC has been used in both metabolomic
and proteomic studies in search of biomarkers.
Increased resolution in HPLC is achieved by
using smaller packing particles (i.e.,

Personnel and Experimental Validation
Any research to be done correctly requires
trained and competent personnel using vali-
dated and proven methods. Therefore, to avoid
any errors, trained personnel should be used in
every aspect of the research from sample collec-
tion, handling, and storage to sample analysis
and results manipulation. Having personnel
trained in every aspect of the biomarker research
project enables a stronger team of scientists with
the capability of troubleshooting any de

ciencies

2 m m) throughout the course of the study.

and UPLC. 37,38 Wilson et al. 37 reported that

UPLC offered signi cant advantages over

conventional reversed-phase HPLC (up to 4,000
psi). It more than doubled the peak capacity,
giving approximately a 10-fold increase in speed
and a three- to

SPECIFICITY OF PROTEINS
AS BIOMARKERS

fivefold increase in sensitivity
compared to that generated with a conventional
3.5

uid
or tissue is like searching for a needle in a
haystack; however, the search may result in
multiple proteins that are each involved in

The search for a protein biomarker in a bio

m stationary phase. Although UPLC/MS/
MS using a single column possesses a high

m