data-dependent acquisition (DDA) mass spectrometry (MS). This
chapter further describes one potential downstream summary analysis of the quantitative proteomic results by term enrichment
analysis.
The proteomic sample preparation workflow has many variations, but they all follow the same skeleton of necessary steps. First,
proteins are isolated from tissues or cultured cells using aqueous
buffer containing a solubilizing and denaturing agent (e.g., guanidine, urea, sodium dodecyl sulfate). Disulfide bonds between cysteine residues in those proteins are reduced and covalently
alkylated. “Shotgun proteomics” refers to the fact that before
proteome analysis proteins are enzymatically hydrolyzed into peptides, which is done because direct analysis of intact proteins is
analytically challenging compared to peptides. Usually, trypsin is
used to catalyze peptide production because trypsin’s specificity for
arginine and lysine residues produces peptides with good length
and charge character for MS detection. Finally, peptides produced
from enzymatic hydrolysis are purified from interfering substances
(e.g., buffer, denaturant, undigested protein) using solid phase
extraction. This highly complex mixture of purified peptides is
ready for shotgun proteomic analysis.
Peptides are most commonly analyzed by liquid chromatography (LC) coupled to a hybrid mass spectrometer capable of isolating intact ions and fragmenting them before measuring fragment
masses, which is a process called tandem mass spectrometry
(MS/MS). There are many ways to operate a hybrid mass spectrometer for peptide analysis. One common method for data collection is data-dependent acquisition, in which the mass
spectrometer surveys all peptide masses eluting from the LC column at every point in time (precursor ion scan or MS1 scan) and
then selects a number of the most abundant masses to be isolated
for fragmentation by collision with an inert gas. The peptide fragment masses resulting from peptide degradation in the gas phase
are measured to produce a discreet snapshot called a tandem mass
spectrum (or MS/MS spectra). Many thousands of MS/MS spectra
are collected from each LC-MS/MS experiment, which are used for
qualitative analysis of peptides in a sample.
After tandem mass spectrometry data are collected, the first
step in data analysis is to identify peptides and proteins (Fig. 1a).
First, the raw data files produced by the mass spectrometer store
data in proprietary formats that must be converted to an open
format before subsequent analysis. Most commonly, the raw data
are converted to “mzXML” or “mzML” formats, which are
human-readable XML. Those files are then used to generate peptide identifications. Peptides are matched to tandem mass spectra
by a process called “database search” to produce peptide-spectra
matches (PSMs). Peptide sequences are predicted from the organism’s genome sequence, and the theoretical fragmentation patterns
298
Jesse G. Meyer
chapter further describes one potential downstream summary analysis of the quantitative proteomic results by term enrichment
analysis.
The proteomic sample preparation workflow has many variations, but they all follow the same skeleton of necessary steps. First,
proteins are isolated from tissues or cultured cells using aqueous
buffer containing a solubilizing and denaturing agent (e.g., guanidine, urea, sodium dodecyl sulfate). Disulfide bonds between cysteine residues in those proteins are reduced and covalently
alkylated. “Shotgun proteomics” refers to the fact that before
proteome analysis proteins are enzymatically hydrolyzed into peptides, which is done because direct analysis of intact proteins is
analytically challenging compared to peptides. Usually, trypsin is
used to catalyze peptide production because trypsin’s specificity for
arginine and lysine residues produces peptides with good length
and charge character for MS detection. Finally, peptides produced
from enzymatic hydrolysis are purified from interfering substances
(e.g., buffer, denaturant, undigested protein) using solid phase
extraction. This highly complex mixture of purified peptides is
ready for shotgun proteomic analysis.
Peptides are most commonly analyzed by liquid chromatography (LC) coupled to a hybrid mass spectrometer capable of isolating intact ions and fragmenting them before measuring fragment
masses, which is a process called tandem mass spectrometry
(MS/MS). There are many ways to operate a hybrid mass spectrometer for peptide analysis. One common method for data collection is data-dependent acquisition, in which the mass
spectrometer surveys all peptide masses eluting from the LC column at every point in time (precursor ion scan or MS1 scan) and
then selects a number of the most abundant masses to be isolated
for fragmentation by collision with an inert gas. The peptide fragment masses resulting from peptide degradation in the gas phase
are measured to produce a discreet snapshot called a tandem mass
spectrum (or MS/MS spectra). Many thousands of MS/MS spectra
are collected from each LC-MS/MS experiment, which are used for
qualitative analysis of peptides in a sample.
After tandem mass spectrometry data are collected, the first
step in data analysis is to identify peptides and proteins (Fig. 1a).
First, the raw data files produced by the mass spectrometer store
data in proprietary formats that must be converted to an open
format before subsequent analysis. Most commonly, the raw data
are converted to “mzXML” or “mzML” formats, which are
human-readable XML. Those files are then used to generate peptide identifications. Peptides are matched to tandem mass spectra
by a process called “database search” to produce peptide-spectra
matches (PSMs). Peptide sequences are predicted from the organism’s genome sequence, and the theoretical fragmentation patterns
298
Jesse G. Meyer