Proteomic and Mass Spectrometry Technologies for Biomarker Discovery - Proteomic and Metabolomic Approaches to Biomarker Discovery - page 23

intact proteins, referred to as top-down proteo-
mics, or identi

cation-exchange and reversed-phase chromatog-
raphy, andwell ionized by electrospray ionization
(ESI). To increase the number of peptide identi

cation of protein cleavage prod-
ucts, referred to as bottom-up or shotgun
proteomics. Top-down strategies retain a lot of
information about protein sequence, protein iso-
forms, as well as their PTMs. Recent advances in
top-down proteomics allow for identi

ca-
tions and protein sequence coverage, protein
digestion protocols may be complemented by
proteases with different sequence speci cities,

such as LysC, ArgC, AspN, and GluC. 43

cation of
hundreds of intact proteins in yeast and
mammalian cells 40,41 ; however, clinical applica-
tions of top-down proteomics are still limited.
Bottom-up proteomic approaches suffer from
a loss of information about protein isoforms and
PTMs, especially for low-abundance proteins.
On the contrary, bottom-up proteomics greatly
bene

Protein and Peptide Separation
Techniques
In the last two decades, two-dimensional poly-
acrylamide gel electrophoresis was a method of
choice for protein separation in both top-down
and bottom-up protein identi

ts from superior liquid chromatography
(LC) separation of peptides prior to mass spec-
trometry, requires lower amounts of material,
and provides better peptide fragmentation and
higher sensitivity. Due to the very high number
of routine protein identi

cation work-

ows. 44,45 Lately, with advances of bottom-up
proteomics, separation techniques focused on
fractionation of short tryptic peptides. Work-

flows with two-dimensional separation of
peptides by strong cation-exchange chroma-
tography or isoelectric focusing followed by
reversed-phase liquid-chromatography allowed
for identi

cations in biological
samples, bottom-up proteomics remains the
platform of choice for biomarker discovery
pipelines. Process of protein identi cation by

bottom-up proteomic methods involves a set of
consecutive steps, such as protein digestion,
peptide separation by LC, peptide ionization,
gas-phase peptide separation, peptide fragmen-
tation, and detection of mass-to-charge ratios
(m/z) and intensities of peptide ions and their
tandem mass spectrometry (MS/MS) fragments.
The variety of mass spectrometry platforms used
for protein identi

cation of thousands of proteins and

cant increase in proteome coverage. 46,47 signi

Protein and Peptide Ionization
Techniques
One of the mass spectrometry advancements
that facilitated routine identi cation of proteins

and peptides in biological samples was the
discovery of soft ionization techniques. 48,49 Soft
ionization such as matrix-assisted laser desorp-
tion ionization (MALDI) 48 and ESI 49 improved
the transfer of large biological molecules into
the gas phase without signi

cation is described in the
following subsections.

Protein Digestion
Bottom-up proteomic approaches involve
proteolytic cleavage of proteins into short peptide
fragments by proteases. The most widely used
enzyme is a chemically modi

cant structural

decomposition.
MALDI-MS (mass spectrometry) is applied to
the analysis of a variety of molecules that range
from small organic compounds to large biomole-
cules such as immunoglobulins. 50 MALDI is initi-
ated by the absorption of a UV laser beam by
a matrix material mixed with a biological
sample. 48 As the laser strikes the matrix, it causes
ablation of

ed trypsin that
selectively cleaves peptide bonds C-terminal to
lysine and arginine residues. 42 A distinct advan-
tage of the use of trypsin is generation of
short doubly or triply charged peptides that are
water soluble, well separated by both strong the surface material and the