Sample Depletion, Fractionation, and Enrichment for Biomarker Discovery - Proteomic and Metabolomic Approaches to Biomarker Discovery - page 231

proteome is made up of tens of thousands of poly-
peptides/proteins that possess the same amino
acid building blocks; however, they vary in size
and cover a wide range of physical and chemical
properties (e.g., size, acidity, basicity, hydropho-
bicity, hydrophilicity, etc.). Unlike the proteome,
the metabolome contains compounds that are
structurally unrelated but also possess awide range
of different chemical and physical properties.
Metabolites such as carbohydrates, salts, lipids,
steroid hormones, hydrocarbons, and so on often
require different procedures for enrichment, extrac-
tion, fractionation, separation, and detection.
Sample fractionation, a procedure for simpli-
fying complex mixtures, is an important step in
the search for biomarkers in clinical samples.
Fractionation of a sample is different fromaliquot-
ing a sample. Aliquoting is a simple procedure in
which the sample (blood, urine) is divided into
smaller portions (aliquots); fractionation is the
division of the sample

lower-abundance species. Examples of depletion
steps include the removal of human serum
albumin from blood (serum and plasma) and
salts and lipids from urine. The bene

ts of deple-
tion include: (a) the detection of low-abundance
proteins and low-level metabolites that are
masked by the presence of the depleted
compounds, (b) the disruption of the binding of
metabolites to proteins, and (c) minimization of
ionization suppression when using mass spec-
trometry (MS) as a detection device. The protein
content of blood (serum, plasma) is dominated
by high-abundance proteins (albumin,

trans-

ferrin, a -1 acid glycoprotein, fibrinogen, cerulo-

plasmin, 1-antitrypsin,

apolipoprotein, plasminogen, haptoglobin, and
prealbumin) that constitute 99% of the protein
content of blood ( Figure 1 ). Therefore, only 1%
of the entire blood proteome is made up of
proteins that are considered to be in low abun-
dance and of particular interest in the search for
potential biomarkers. Therefore, depletion of the
high-abundance proteins is necessary for the
detection of the low-abundance proteins in blood.
The most popular methods for depleting albumin
and other high-abundance proteins from blood
include immunoaf

2-macroglobulin,

a a

' s “ contents ” (peptides,

proteins, metabolites) into several (frac-

tions) based on their chemical and physical prop-
erties, generally using chromatography or
electrophoresis. Extraction, unlike depletion, is
a puri

“ groups ”

cation step with the aim of selecting and
enriching a single or group of compounds of
interest from a mixture, such as extraction of
estrogen metabolites from urine or serum. The
purpose of extraction, depletion, and fraction-
ation in proteomic and metabolomic analysis is
to simplify the complexity of the mixture and
allow the detection of low-abundance proteins
and metabolites, resulting in an increase in the
number of compounds that can be identi

nity columns or membrane

filters.1 1
Analysis of the metabolome of biological
samples such as urine and serum requires the
precipitation of proteins followed by the analysis
of the supernatant. Precipitation of proteins can
be achieved by heat or the addition of an acid
or organic solvent such as acetonitrile, ethanol,
or methanol. Removal of salts, lipids, and so on
from the sample can be accomplished by column
chromatography,

ed.

solid phase extraction, or

DEPLETION liquid liquid extraction.

e

Depletion is an essential step in the analysis
of the proteome and metabolome. It is a process
for the removal of proteins and other compounds
from the sample that can interfere with the
analytical procedure and affect

FRACTIONATION PROCEDURES
FOR PROTEINS AND METABOL ITES

the accuracy Fractionation of protein mixtures prior to

separation and MS analysis can be carried out of the results or the ability to detect