Tissue Sample Preparation for Proteomic Analysis - Proteomic and Metabolomic Approaches to Biomarker Discovery - page 40

fresh-frozen (FF) tissues and formalin- xed are issues that should be considered before

designing any MS-based biomarker study.

paraf n-embedded (FFPE) tissues. A compar-

ison of the suitability of these two tissue types
for proteomic analysis is shown in Table 1 .

Formalin-Fixed Paraf n-Embedded

Tissue
Although FF tissue remains the gold standard
for extraction and large-scale pro

Fresh and/or Fresh-Frozen Tissue
Fresh or FF tissue is the specimen of choice for
MS-based proteomics because proteins and
other biomolecules (i.e., metabolites, nucleic
acids) are unmodi

ling of proteins
and other biomolecules in vivo , 11 there is an
increasing interest in analyzing the protein
complement of FFPE tissues. 12 This interest is
primarily driven by the fact that the process of
creating FFPE tissues is the most common
technique for tissue processing, evaluation, diag-
nostics, immunoanalysis, preservation, and
archiving used by clinical and/or research
pathologists. It is estimated that worldwide,
more than a billion FFPE tissue samples are
stored in hospitals, tissue banks, and research
laboratories. 13 These archived FFPE tissues may
provide a wealth of information when used in
retrospective molecular

ed and preserved in their
natural tissue environment. The most commonly
used method is snap-freezing in liquid nitrogen
(LN 2 ). It provides excellent protein integrity
and a wide array of options for MS-based pro-
teomic pro ling. Ideally, any tissue specimen

should be snap-frozen within five minutes after

surgical removal, biopsy, or laboratory dissec-
tion. Upon acquisition, fresh tissue is placed
into a sterile cryovial, which is then tightly
capped and submerged in LN 2 for snap-
freezing. This vial is transferred from the tempo-
rary LN 2 transport container into a long-term
liquid nitrogen storage tank or in a freezer
at e 80 C. Fresh-frozen specimens are typically
processed and analyzed as tissue blocks, 3,7,8
thin tissue slices, 9 or laser capture microdissec-
tion (LCM) specimens. 10 It is important to note
that the availability and storage of FF tissues

studies focused on

ling and biomarker research. 12
Thus, it is not surprising that signi

molecular pro

cant efforts
have been put into developing methods for
analyzing FFPE, exempli

ed by increasing
number of studies using this tissue type
for biomarker discovery by MS-based
proteomics. 14 e 18 Tissue specimens intended for
formalin

n embedding should
be trimmed to be approximately 0.5-cm-thick
blocks. The tissue should be

fixation and paraf

TABLE 1 Suitability of FF and FFPETissues forMolecular
Pro ling and Biomarker Development Using
MS-Based Proteomics

xed at room
temperature for a minimum of 24 hours, at which
point the tissue can be transferred to a histology
lab for embedding in paraf

FF FFPE

in Similar to FF
tissue, FFPE tissue specimens are typically
received and processed as tissue blocks, 19 thin
tissue slices, 16 or LCM specimens. 18,20 However,
high-throughput proteomic studies using FFPE
tissues have been challenging, primarily because
of formaldehydeinduced protein crosslinks
during

Availability D DDD

Storage costs DDD D

Protein accessibility DDD DD

Protein quality DDD D

MS-based quantitation
accuracy

DDD DD

fixation process. Therefore, a reversal of
the formaldehyde induced crosslinks and
protein adducts is mandatory for extracting
intact proteins prior to fractionation and/or

DDD D

Protein immunoreactivity

Sample preparation costs DD DDD