Tissue Sample Preparation for Proteomic Analysis - Proteomic and Metabolomic Approaches to Biomarker Discovery - page 43

(e.g., Barocycler , Pressure Biosciences Inc.)
that employs alternating hydrostatic pressure
in combination with mechanical grinding to
disrupt tissue specimens. 29 This technique desta-
bilizes intermolecular interactions by rapidly
and repeatedly raising and lowering pressure
in the reaction vessel from ambient to high levels
( 35,000 psi). It utilizes compressed air to create

high and ultra-high hydrostatic pressure inside
the Barocycler reaction chamber. This tech-
nology has been employed to isolate intact and
functional mitochondria from muscle,

w

liver,

and kidney tissue. 30

Ultrasonic Homogenizers
Ultrasonic homogenizers utilize sound waves
to disrupt tissues (e.g., Model 250 Digital

Soni er , Branson Ultrasonics Corp.). Tissue

ultrasonic disruptors are typically equipped
with a stainless-steel-tip generator. By placing
the end of the tip into the extraction buffer,
the tip moves the liquid forward (away) and
then retracts (back) faster than the liquid can
return, resulting in the cavitation effect capable
of disrupting the tissue. Ultrasonic homoge-
nizers are effective in homogenizing small
pieces of soft tissues (e.g., epidermis). 31
However, they are not suitable for tough and
dense tissues.

FIGURE 2 (A) Mini bead beater, (B) bead-beater mill. 38

that can handle multiple samples simulta-
neously (e.g., Bead Ruptor , Omni Interna-
tional). This bead beater can handle from 4 to
24 tissue samples simultaneously ( Figure 2 B). It
is speci

EXTRACTION/SOLUBILIZATION
BUFFERS

cally designed for grinding and homog-
enizing biological samples prior to molecular
extraction. The disposable vials are pre

Tissue homogenization represents a critical
step in the proteomic work

lled, by
the manufacturer, with beads of different sizes
(1.4 to 2.8 mm) and properties (ceramic, glass,
or metal) depending on the type of tissue (soft
or hard). Unfortunately, protein alterations
may happen due to overheating or wear of the
grinding media.

ow for produc-
ing reproducible protein extraction methods.
Ideally, for effective bottom-up tissue proteo-
mics, the extraction buffer should provide
optimal conditions for protein extraction, solubi-
lization, and subsequent proteolysis. To mini-
mize unnecessary tissue manipulations and
potential sample losses, it is important that
the buffer components (e.g., ionic detergents,
chaotropes, salt, etc.) do not

Pressure Cycling Homogenizers
Pressure cycling technology (PCT) is a pres-
sure-mediated tissue homogenization technique interfere with