Tissue Sample Preparation for Proteomic Analysis - Proteomic and Metabolomic Approaches to Biomarker Discovery - page 41

tryptic digestion. Numerous methods have
been developed to facilitate the retrieval of
intact proteins from FFPE tissues. Almost all
published methods rely on a variation of the
heat-induced antigen retrieval technique 21 in
which the FFPE tissue is exposed to an
elevated temperature of about 90 C at normal
or elevated pressure for a period of about 30
minutes. Subsequently, proteins are solubilized
and digested using detergents, 22 a combination
of detergents and chaotropes, 18

minimal mechanical force. It is mandatory to
perform tissue homogenization at low tempera-
tures (i.e., 4 C) to minimize endogenous enzyme
activity. It is not uncommon to use multiple tech-
niques in combination. In general, the homogeni-
zation of FF tissues can be done using manual
tools or commercially available apparatuses.

Manual Tools for Tissue
Homogenization
The commonly used manual tools for tissue
homogenization rely on mechanical pressure.
These simple and affordable tools disrupt tissues
when a moving pestle pinches the samples
against the wall of the mortar. The choice of
a tool is driven by physical characteristics of
the tissue being analyzed and the number of
samples to be processed. The most common
tools are glass (e.g., Potter-Elvehjem tissue
homogenizer, Dounce tissue homogenizer), Tef-
lon (e.g., liquid-nitrogen-cooled mortar and
pestle tissue grinders), and stainless steel (e.g.,
freeze-fracturing tissue pulverizers).

or organic

solvents. 16,19
Regardless of the extraction ef

ciency, the
quantity and quality of proteins, nucleic acids,
and metabolites obtained from FFPE tissues are
inferior to those obtained from FF tissues. 11,23
However, the availability of FFPE tissues and
recent improvement in removal of protein cross-
links make FFPE an attractive specimen for
MS-based proteomics.

TISSUE DISRUPTION/
HOMOGENIZATION

Glass Homogenizers/Grinders
Potter-Elvehjem and Dounce types are the
most commonly used glass homogenizers for
disruption of soft tissues (e.g., Potter-Elvehjem
homogenizer, Thomas Scienti

Tissue specimens must be handled and
prepared in a standardized fashion to provide
reproducible results. Immediately upon acquisi-
tion, fresh tissue is frozen by quenching in LN 2 .
Tissue preparation for MS-based proteomics
starts with homogenization optimized according
to the physical characteristics of the tissue being
analyzed. The choice of the homogenization
method is crucial because it affects the extent/
coverage of protein identi

c, Swedesboro,
NJ, USA). 7 Using the mechanical shear force
generated by a glass pestle, these tools rapidly
release biomolecules from intracellular and
extracellular compartments into a selected buffer
and pose little risk to proteins other than the
release of proteases from cellular compartments.
Proteolytic degradation can be minimized by the
inclusion of protease inhibitors in the homogeni-
zation buffers.

cation and biological
interpretation of the data. Tissues that are diffi- -

cult to homogenize include bone, heart muscle,
lung, intestine, and skin. For these tissues, an
increase of mechanical force or time of exposure
signi

ciency.
However, the use of excessive force should be
limited because of the generation of excessive
heat and/or shear that can ruin the desired
proteins. On the other hand, soft tissues (e.g.,
brain, liver, spleen) can be homogenized using

cantly improves disruption ef

Glass-Teflon Homogenizers/Grinders
The most on

homogenizer is the Potter-type (e.g., Pestle
Tissue Grinder 1ml, Thomas Scienti

commonly used glass-Te

c, NJ,

USA). Te on, a synthetic fluoropolymer of