Tissue Sample Preparation for Proteomic Analysis - Proteomic and Metabolomic Approaches to Biomarker Discovery - page 44

protein or peptide separation and downstream
MS analysis. Typically, proteins and peptides
extracted from tissue homogenates are resolved
and analyzed using gel-based or gel-free
LC-MS approaches.

outcome. These findings were cross-validated

by Western blotting,

rming Gal-1 as
a potential prognostic and predictive biomarker
for cHL.
Another 2D-PAGE-based study, by Junker
et al., was focused on identi

con

cation of potential
progression markers in kidney cancer. 33
Tumorous and adjacent normal tissue were
homogenized using mechanical grinder in the
presence of the TRIzol Reagent (Invitrogen,
Karlsruhe, Germany). Homogenates represent-
ing three different tumor stages (pT1, pT2, pT3)
were resolved using two-dimensional difference
gel electrophoresis (2D-DIGE). Selected spots
that showed an abundance difference between
the tumor stages were in-gel digested and
identi

Buffers Used in Gel-Based Tissue
Proteomics
The principal active constituent of buffers
used in gel-based proteomics is a detergent, typi-
cally sodium dodecyl sulfate (SDS). It denatures
and solubilizes proteins for analysis using 1D- or
2D-PAGE. Despite the low resolving capacity of
the 1D-PAGE and 2D-PAGE

s inability to
resolve membrane proteins, proteins with
extreme MWs and/or pIs, these approaches
have been extremely useful for tissue molecular
pro

'

ed by MALDI-TOF-MS/MS. On the
2D-DIGE protein map, out of 989 visualized
spots a total of 176 were determined as at least
twofold differentially expressed. This study
generated a kidney cancer molecular phenotype
map that revealed stage-related alterations in
protein abundance. Ingenuity Pathway Analysis
was used to explore direct networks connections
between all identi

ling and biomarker research.
A study by Kemper et al. showed the utility
of the SDS extraction and solubilization buffer
coupled with

2D-PAGE for comparative

pro ling of FF tissues obtained from patients

diagnosed with classic Hodgkin lymphoma
(cHL). 32 The aim of this study was to identify
possible prognostic protein markers in advanced
stages of cHL. A total of 14 cHL pretreatment
tissue samples from younger patients with
advanced-stage disease were analyzed. Patients
were grouped according to treatment responses.
A total of 70 tissue sections of 20

ed proteins showing altered
expression. The most signi cantly activated

networks included embryonic development,
lipid metabolism, and small molecule biochem-
istry. After hierarchical clustering and principal
component analysis, three candidate protein
markers (i.e., prohibitin, thioredoxin-dependent
peroxide reductase PRDX3 and S100-A9) were
selected and cross-validated by immunohisto-
chemistry and Western blotting. Cross-
validated proteins might be the starting point
for further research towards better under-
standing of kidney tumor development and
development of diagnostic or prognostic protein
biomarkers.

m were
loaded into empty Eppendorf tubes and homog-
enized using a mechanical grinder in the pres-
ence of a lysis buffer containing 9M urea, 2%
(wt/vol) Triton X-100, 2% (wt/vol) DTT, and
2% (vol/wt) IPG-buffer (Amersham). Differen-
tially expressed proteins between the groups
were separated using 2D-PAGE and identi

m

ed
by LC-MS. The comparison revealed upregula-
tion of the immunoregulatory glycan-binding
protein Gal-1 in patients with poorer outcome;
PRDX1, a member of a family of antioxidant
enzymes, and GNAI3, a cell migration e associ-
ated protein, were found to be substantially
upregulated in patients with a

Buffers Used in Gel-Free Tissue
Proteomics
Due to the limitations of gel-based proteo-
mics, favorable the gel-free “ shotgun ” proteomics is