Tissue Sample Preparation for Proteomic Analysis - Proteomic and Metabolomic Approaches to Biomarker Discovery - page 46

hydrophobic membrane proteins within tissue
homogenates. 38

study showed that DTP is amenable to ef ciently

identify proteins from FF and FFPE tissues.
Approximately twice the numbers of proteins
were identi

Aqueous/Organic Solvents e Based Buffers
Aqueous/organic buffers are LC/MS compat-
ible while facilitating protein solubilization,
extraction, and denaturation, and alleviate
digestion. 39 e 42 To improve molecular pro

ed from the FF sections when
compared with the FFPE sections. The distinct
expression of biologically relevant proteins was
cross-validated by immunohistochemistry. This
study provided the

ling first large-scale proteomics

map of human coronary atherosclerotic plaques.
Characterization of disease-relevant molec-
ular phenotypes from limited tissue specimens,
such as pathological biopsies and tissues from
small model organisms, remains an analytical
challenge and a much-needed clinical goal. We
used a transgenic mouse model of cardiac-
speci

of tissue homogenates and avoid the de ciencies

of traditional approaches, a method that relies on
60% buffered methanol to extract, solubilize, and
digest plasma membrane proteins isolated from
human skin was developed. 7,43
Subsequently, an extra step was added to the
original protocol for solubilization and digestion
of proteins in tissue homogenates obtained by
LCM from FF breast cancer tissue and stroma,
respectively. 10 In this two-stage protocol, 20%
buffered methanol was added to facilitate diges-
tion of cytosolic proteins while 60% buffered
methanol was used to facilitate solubilization
and digestion of hydrophobic/membrane
proteins. This two-stage procedure was also
used in a tissue-directed biomarker discovery
proof of principle study using renal cell carcinoma
(RCC) as a model disease, 3 as well as in a study
focused on comparative molecular pro

c H-Ras-G12V-induced hypertrophic
cardiomyopathy to explore the potential of using
MS-based proteomics to obtain a disease-

relevant molecular pro le from amount-limited

FF tissue specimens routinely used in patholog-
ical diagnosis. 9 The described method relies on
two-stage methanol-assisted extraction and solu-
bilization

to digest protein homogenates

prepared from 8- m-thick FF histological tissue

sections of diseased/experimental and normal/
control hearts. The 8-

m

m tissue sections were
scraped from the slides, transferred to Eppen-
dorf tubes, and homogenized using tip sonica-
tion. 9 The disease-driving H-Ras protein and
vimentin were unambiguously identi

m

ling of
H-Ras-G12V-induced hypertrophic cardiomyop-
athy in transgenic mice using LC-MS analysis of
thin FF tissue sections. 9 Differently formulated
aqueous and organic buffers were also used in
studies analyzing FFPE tissues obtained from
prostate, colon, and uterus, respectively. 16,19,20
In their study, Bagnato et al. compared molec-
ular phenotypes of normal and diseased arteries
using proteomic analysis of FFPE tissues
acquired by LCM to dissect and capture the
intima, the tunica media, and the tunica adven-
titia. 23 After sonication, protein complement of
LCM-acquired specimens was homogenized,
extracted, solubilized, and digested in organic/
aqueous buffer using previously developed
direct tissue proteomics (DTP) methodology
focused on the analysis of thin prostate FFPE
tissue slices. 16 The

ed by
LC-MS in hypertrophic myocardium and cross-
validated by immunohistochemistry and

Western blotting. The pathway analysis

involving proteins identi ed by MS showed

strong association of proteomic data with cardio-
vascular disease. More importantly,

the MS

identi cation and subsequent cross-validation

of Wnt3a and

b -catenin, in conjunction with

IHC identi cation of phosphorylated GSK-3 b

and nuclear localization of b -catenin, provided

evidence of Wnt/ -catenin canonical pathway

activation secondary to H-Ras activation in the
course of pathogenic myocardial hypertrophic
transformation. The results of this study show
the

b

findings from this feasibility capability of described approach for