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increasingly used in biomarker research via
molecular pro
isomerase A 4 (PDIA4) were found to be upregu-
lated in ESCC and were subsequently validated
by immunohistochemical labeling using tissue
microarrays.
Proteomic analysis of clinical tissue speci-
mens isolated by LCM requires sample prepa-
ration and fractionation methods suitable for
limited amounts of sample. Wisniewski et al.
showed that the combination of 4% SDS and
8 M urea effectively solubilizes proteins from
archival neoplastic and matched normal colonic
cancer specimens.
31
Specimens obtained by
LCM from three patients allowed ef
ling of a large series of clinical
specimens. Bottom-up shotgun proteomics typi-
cally utilizes multiple dimensions of LC separa-
tion to reduce sample complexity prior to MS
analysis of resolved peptide fractions.
34
The
most commonly used fractionation technique
employs strong cation exchange (SCX) LC to
resolve/fractionate tissue digestates.
35
Detergent- and Chaotrope-Based Buffers
Used in Gel-Free Tissue Proteomics
It is well accepted that detergents and chao-
tropes interfere with digestion, separation, or
ionization of proteins and peptides and may
compromise MS analysis.
36
However, due to
recent improvements in their removal, deter-
gents alone
4
or in combination with chaotropes
have been increasingly used to extract, solubi-
lize, and digest protein complement in tissue
homogenates prior
cient anal-
ysis of tissue extracts, containing low numbers
(i.e., 500, 1,000, and 3,000) of cells, using
a streamlined
filter-aided sample preparation
ow.
37
Incorporation of an addi-
tional micro SCX fractionation step enabled
the analysis of FFPE tissues prepared by
LCM to a depth of 3,600 to 4,400 proteins per
single experiment. The analysis resulted in the
identi
(FASP) work
to fractionation and or
LC-MS analysis.
18
In a study focused on the identi
cation of 30 known colon cancer
markers. These included carcinoembryonic
antigen (CEA; the most widely used colon
cancer marker), complement decay accelerating
factor (DAF, CD55), and metastasis-associated
in colon cancer protein 1 (MACC1). Impor-
tantly, mucin 1 was found overexpressed and
mucin 2 underexpressed in all three patients.
These results show that the extraction buffer
containing high concentration of SDS and
urea within the FASP work
cation of
potential biomarkers for esophageal squamous
cell carcinoma (ESCC), Pawar et al. employed
a 0.5% SDS-based buffer to solubilize and digest
proteins for pro
ling of respective tissue homog-
enates using isobaric tags (i.e., iTRAQ).
4
Tissue
specimens were homogenized using cell
disperser (IKA works, Wilmington, NC, USA).
Protein expression pro
les in ESCC tumor
tissues were compared with the corresponding
adjacent normal tissue specimens obtained
from ten patients. After SCX-based fractionation,
LC-MS/MS analysis led to the identi
ow is suitable for
in-depth analysis of LCM tissue and has the
potential
for biomarker and drug target
cation of
687 proteins. A total of 257 proteins were found
differentially expressed in ESCC compared to
normal tissue. Several previously known protein
biomarkers were found to be upregulated in
ESCC including thrombospondin 1 (THBS1),
periostin 1 (POSTN), and heat shock 70 kDa
protein 9 (HSPA9). In addition, several novel
proteins were identi
discovery.
The results of these studies indicate that the
MS interfering compounds, detergents (i.e.,
SDS) alone or in combination with chaotropes
(i.e., urea), when ef
ciently removed prior to
LC-MS analysis, do not signi
cantly interfere
with the outcome of LC-MS analyses. However,
the exclusive use of chaotropes (i.e., urea) for
extraction and solubilization of proteins from
tissue specimens is not recommended since
urea alone
ed in this study. These
novel biomarker candidates: prosaposin
(PSAP), plectin 1 (PLEC1), and protein disul
de
is not
capable of
solubilizing
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