67. Load the sample by pipetting the protein digest up and down
(discarding it back into its tube). Repeat at least 10Â.
68. Elute the peptides by pipetting the ZipTip up and down in the
elution buffer (discarding it back into its tube). Repeat at least
10Â.
69. Ensure there are no air bubbles at the bottom of the vial.
70. Equilibrate the column for at least 15 min.
71. Tuning if necessary and save the new tune method after the
successful tuning.
72. Inject 1 μL of the 500 fmols/μL cyt c standard solution per
four sample injections and use cyt c solution as LC–MS/MS
data quality control.
73. We work with the linear ion trap mass spectrometer LTQ Velos
Pro with electrospray ionization (ESI). Adjust the MS/MS
method if you work with any other mass spectrometer.
74. Our search criteria for the identification of carbonylated proteins are: oxidation of methionine and carbamidomethylation
of cysteine as variable modifications, trypsin as the proteolytic
enzyme, maximum 2 missed cleavages per peptide and a mass
tolerance of Æ1 Da for precursor and Æ0.6 Da for product ion
scans. False discovery rate (FDR) for identifications were
accepted if <1%. Modify the search parameters according to
the properties of the mass spectrometer used.
Acknowledgments
This work was supported by the Spanish Ministry of Science and
Innovation (grant RTI2018-095659-B-I00). Xunta de Galicia and
Axencia Galega de Innovacio ´ n (GAIN) are thankfully recognized
by the financial support of the postdoctoral contract to Lucı ´a
Me ´ndez (IN606B 2017/006). The Spanish Ministry of Science
and Innovation is also gratefully acknowledged for the doctoral
fellowship to Silvia Mun ˜oz (BES-2014-070757).
References
1. Fedorova M, Bollineni RC, Hoffmann R (2014)
Protein carbonylation as a major hallmark of
oxidative damage: update of analytical strategies.
Mass Spectrom Rev 33:79–97
2. Madian AG, Myracle AD, Diaz-Maldonado N
et al (2011) Differential carbonylation of proteins as a function of in vivo oxidative stress. J
Proteome Res 10:3959–3972
3. Me ´ndez L, Mun ˜oz S, Miralles-Pe ´rez B et al
(2020) Modulation of the liver protein
carbonylome by the combined effect of marine
omega-3 PUFAs and grape polyphenols supplementation in rats fed an obesogenic high fat and
high sucrose diet. Mar Drugs 18:1–30
4. Dalle-Donne I, Giustarini D, Colombo R et al
(2003) Protein carbonylation in human diseases.
Trends Mol Med 9:169–176
5. Chaudhuri AR, de Waal EM, Pierce A et al
(2006) Detection of protein carbonyls in aging
Fluorescent 2DE-LC-MS for Carbonylome Analysis
245
(discarding it back into its tube). Repeat at least 10Â.
68. Elute the peptides by pipetting the ZipTip up and down in the
elution buffer (discarding it back into its tube). Repeat at least
10Â.
69. Ensure there are no air bubbles at the bottom of the vial.
70. Equilibrate the column for at least 15 min.
71. Tuning if necessary and save the new tune method after the
successful tuning.
72. Inject 1 μL of the 500 fmols/μL cyt c standard solution per
four sample injections and use cyt c solution as LC–MS/MS
data quality control.
73. We work with the linear ion trap mass spectrometer LTQ Velos
Pro with electrospray ionization (ESI). Adjust the MS/MS
method if you work with any other mass spectrometer.
74. Our search criteria for the identification of carbonylated proteins are: oxidation of methionine and carbamidomethylation
of cysteine as variable modifications, trypsin as the proteolytic
enzyme, maximum 2 missed cleavages per peptide and a mass
tolerance of Æ1 Da for precursor and Æ0.6 Da for product ion
scans. False discovery rate (FDR) for identifications were
accepted if <1%. Modify the search parameters according to
the properties of the mass spectrometer used.
Acknowledgments
This work was supported by the Spanish Ministry of Science and
Innovation (grant RTI2018-095659-B-I00). Xunta de Galicia and
Axencia Galega de Innovacio ´ n (GAIN) are thankfully recognized
by the financial support of the postdoctoral contract to Lucı ´a
Me ´ndez (IN606B 2017/006). The Spanish Ministry of Science
and Innovation is also gratefully acknowledged for the doctoral
fellowship to Silvia Mun ˜oz (BES-2014-070757).
References
1. Fedorova M, Bollineni RC, Hoffmann R (2014)
Protein carbonylation as a major hallmark of
oxidative damage: update of analytical strategies.
Mass Spectrom Rev 33:79–97
2. Madian AG, Myracle AD, Diaz-Maldonado N
et al (2011) Differential carbonylation of proteins as a function of in vivo oxidative stress. J
Proteome Res 10:3959–3972
3. Me ´ndez L, Mun ˜oz S, Miralles-Pe ´rez B et al
(2020) Modulation of the liver protein
carbonylome by the combined effect of marine
omega-3 PUFAs and grape polyphenols supplementation in rats fed an obesogenic high fat and
high sucrose diet. Mar Drugs 18:1–30
4. Dalle-Donne I, Giustarini D, Colombo R et al
(2003) Protein carbonylation in human diseases.
Trends Mol Med 9:169–176
5. Chaudhuri AR, de Waal EM, Pierce A et al
(2006) Detection of protein carbonyls in aging
Fluorescent 2DE-LC-MS for Carbonylome Analysis
245