establishing diagnostic and prognostic molecular signatures or biomarkers for different diseases and stratifying individuals by symptoms or response to treatment [3, 4].
High-throughput shotgun proteomics, a powerful tool for the
investigation of multifactorial diseases, can be employed to study
these topics as it can identify and quantify the proteins that are
present in samples at a certain point of time [5, 6]. Such broad
acquisitions are able to reveal possible alterations that cause—or are
a result of—diseases or treatments. Furthermore, molecular signatures or biomarkers can be proposed and confirmed by proteomic
investigations using noninvasive samples such as blood, along with
other tissues [6, 7].
Human blood is relatively easy to obtain. Proteins present in
human blood plasma or serum range over ten orders of magnitude
[8–11]. Furthermore, these proteins may have the potential for
diagnosis due to the direct contact between blood and all internal
organs [10, 11]. Complicating analyses, however, is the fact that
99% of the total plasma protein mass consists of well-known plasma
or serum components—considered the high-abundance fraction—
which can impair the visualization of the low-abundance protein
fraction [8, 12]. The low-abundance fraction is composed of many
proteins with relatively low concentrations and is an important
source of potential biomarkers. Thus, the depletion of the major
proteins of the plasma/serum (as shown in Fig. 1) improves the
detection, identification, and quantitation of low-abundance proteins [13, 14]. Several techniques for the depletion of human blood
plasma exist, but immunoaffinity chromatography fractionation
prior to mass spectrometry analysis represents the state-of-the-art
in this matter, serving to increase the analytical power of sample
analysis [15–18].
When high-performance liquid chromatography (LC) coupled
online with mass spectrometry emerged, it gave rise to shotgun
proteomics, a type of analysis where a mixture of digested proteins—peptides—is separated by reverse-phase chromatography
[19] before their mass is analyzed. Mobile phase gradients allow
molecules present in complex mixtures to be eluted gradually,
improving chromatographic resolution. This fractionation allows
the wide range of peptides present in complex mixtures—including
low-abundant, blood plasma, protein digests—to be separated due
to their different retention times by the stationary phase in the
column. Then, in classic proteomic study design, the peptides are
ionized and conveyed to fragmentation in the mass spectrometer.
The fragmentation is used to acquire information on the massto-charge ratio of ionized precursor ions and the product of fragmentation ions. MS
E mode alternates low and high collision energy
to provide, simultaneously, information about precursor and product ions. An additional dimension of separation for co-eluted peptides can be obtained by using ion mobility separation, a technique
154
Licia C. Silva-Costa et al.
High-throughput shotgun proteomics, a powerful tool for the
investigation of multifactorial diseases, can be employed to study
these topics as it can identify and quantify the proteins that are
present in samples at a certain point of time [5, 6]. Such broad
acquisitions are able to reveal possible alterations that cause—or are
a result of—diseases or treatments. Furthermore, molecular signatures or biomarkers can be proposed and confirmed by proteomic
investigations using noninvasive samples such as blood, along with
other tissues [6, 7].
Human blood is relatively easy to obtain. Proteins present in
human blood plasma or serum range over ten orders of magnitude
[8–11]. Furthermore, these proteins may have the potential for
diagnosis due to the direct contact between blood and all internal
organs [10, 11]. Complicating analyses, however, is the fact that
99% of the total plasma protein mass consists of well-known plasma
or serum components—considered the high-abundance fraction—
which can impair the visualization of the low-abundance protein
fraction [8, 12]. The low-abundance fraction is composed of many
proteins with relatively low concentrations and is an important
source of potential biomarkers. Thus, the depletion of the major
proteins of the plasma/serum (as shown in Fig. 1) improves the
detection, identification, and quantitation of low-abundance proteins [13, 14]. Several techniques for the depletion of human blood
plasma exist, but immunoaffinity chromatography fractionation
prior to mass spectrometry analysis represents the state-of-the-art
in this matter, serving to increase the analytical power of sample
analysis [15–18].
When high-performance liquid chromatography (LC) coupled
online with mass spectrometry emerged, it gave rise to shotgun
proteomics, a type of analysis where a mixture of digested proteins—peptides—is separated by reverse-phase chromatography
[19] before their mass is analyzed. Mobile phase gradients allow
molecules present in complex mixtures to be eluted gradually,
improving chromatographic resolution. This fractionation allows
the wide range of peptides present in complex mixtures—including
low-abundant, blood plasma, protein digests—to be separated due
to their different retention times by the stationary phase in the
column. Then, in classic proteomic study design, the peptides are
ionized and conveyed to fragmentation in the mass spectrometer.
The fragmentation is used to acquire information on the massto-charge ratio of ionized precursor ions and the product of fragmentation ions. MS
E mode alternates low and high collision energy
to provide, simultaneously, information about precursor and product ions. An additional dimension of separation for co-eluted peptides can be obtained by using ion mobility separation, a technique
154
Licia C. Silva-Costa et al.