7. Combine equal volumes (peptides amount) of the labeled
samples in a unique microtube.
8. For peptide fractionation, follow the manufacturer instructions
related to Pierce High pH Reversed-Phase Peptide Fractionation Kit (Thermo Fisher Scientific; Pub. No. MAN0015701)
(see Note 15). Fractionation is based on peptide separation
according to hydrophobicity using a step gradient of increasing
acetonitrile concentrations in a volatile high-pH elution solution yielding eight peptide fractions. Peptide fractions are dried
out using a speed-vacuum concentrator device before longterm storing at À80
C, or for immediate LC–MS/MS analysis
after their resuspension in an appropriate volume (~13 μL) of
0.1% formic acid (see Note 16).
9. Run LC–MS/MS analysis using the higher energy collision
dissociation (HCD) mode (see Note 17) following manufacturer’s instructions [28, 29].
3.7 Mass
Spectrometry Data
Analysis: Protein
Identification
For analysis of raw MS data, set these values for the following
parameters: missed trypsin cleavages ¼ 1, variable modifications ¼ oxidation (M) and deamidation (N, Q), fixed modifications ¼ carbamidomethyl (C) and TMT 10plex (or TMT 6plex,
TMT 11plex) (N-terminal, K) in the case of multiplexed samples.
Mass tolerance for precursor and fragment ions are MS-device
dependent. The values 10 ppm and 0.02 Da for precursor and
fragment ions, respectively, can be taken as a reference value for
data acquired in a Thermo Fisher Scientific LTQ-Orbitrap Elite.
There is a wide list of software including different search engines
(algorithms) for running protein identification and quantification
analysis, some are proprietary (e.g., Mascot, PEAKS, Proteome
Discoverer) while others are free (e.g., PatternLab and MaxQuant).
A full list of mass spectrometry software and diverse MS utilities can
be found here: https://ms-utils.org. In brief, proteomic software
tries first to match experimental MS/MS spectra against theoretical
MS/MS spectra derived from in silico digestion of protein
sequences present in the database (see Subheading 3.8), then peptides and proteins are scored (using a score threshold provides
information about the probability of getting random matches)
according to correspondence between observed and theoretical
fragment ions, and are further filtered out to control for false
discovery rate (FDR) at a predetermined FDR threshold (usually
less than 1%). FDR is empirically calculated by comparing the
proportion of matchings against decoy database out of total matchings (decoy and target protein databases). A decoy database contains a reversed, shuffled, or randomized version of target
sequences, keeping the same size and amino acid frequencies as
the target database, and it is usually created by the software once
the target database has been uploaded [30].
Shotgun Proteomics in Non-model Organisms
89
samples in a unique microtube.
8. For peptide fractionation, follow the manufacturer instructions
related to Pierce High pH Reversed-Phase Peptide Fractionation Kit (Thermo Fisher Scientific; Pub. No. MAN0015701)
(see Note 15). Fractionation is based on peptide separation
according to hydrophobicity using a step gradient of increasing
acetonitrile concentrations in a volatile high-pH elution solution yielding eight peptide fractions. Peptide fractions are dried
out using a speed-vacuum concentrator device before longterm storing at À80
C, or for immediate LC–MS/MS analysis
after their resuspension in an appropriate volume (~13 μL) of
0.1% formic acid (see Note 16).
9. Run LC–MS/MS analysis using the higher energy collision
dissociation (HCD) mode (see Note 17) following manufacturer’s instructions [28, 29].
3.7 Mass
Spectrometry Data
Analysis: Protein
Identification
For analysis of raw MS data, set these values for the following
parameters: missed trypsin cleavages ¼ 1, variable modifications ¼ oxidation (M) and deamidation (N, Q), fixed modifications ¼ carbamidomethyl (C) and TMT 10plex (or TMT 6plex,
TMT 11plex) (N-terminal, K) in the case of multiplexed samples.
Mass tolerance for precursor and fragment ions are MS-device
dependent. The values 10 ppm and 0.02 Da for precursor and
fragment ions, respectively, can be taken as a reference value for
data acquired in a Thermo Fisher Scientific LTQ-Orbitrap Elite.
There is a wide list of software including different search engines
(algorithms) for running protein identification and quantification
analysis, some are proprietary (e.g., Mascot, PEAKS, Proteome
Discoverer) while others are free (e.g., PatternLab and MaxQuant).
A full list of mass spectrometry software and diverse MS utilities can
be found here: https://ms-utils.org. In brief, proteomic software
tries first to match experimental MS/MS spectra against theoretical
MS/MS spectra derived from in silico digestion of protein
sequences present in the database (see Subheading 3.8), then peptides and proteins are scored (using a score threshold provides
information about the probability of getting random matches)
according to correspondence between observed and theoretical
fragment ions, and are further filtered out to control for false
discovery rate (FDR) at a predetermined FDR threshold (usually
less than 1%). FDR is empirically calculated by comparing the
proportion of matchings against decoy database out of total matchings (decoy and target protein databases). A decoy database contains a reversed, shuffled, or randomized version of target
sequences, keeping the same size and amino acid frequencies as
the target database, and it is usually created by the software once
the target database has been uploaded [30].
Shotgun Proteomics in Non-model Organisms
89