Positive Effect of Acetylation on Proteomic Analysis Based on Liquid Chromatography with Atmospheric Pressure Chemical Ionization and Photoionization Mass Spectrometry
A typical bottom-up proteomic workflow comprises sample digestion with trypsin, separation of the hydrolysate using reversed-phase HPLC, and detection of peptides via electrospray ionization (ESI) tandem mass spectrometry. Despite the advantages and wide usage of protein identification and quantific...
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-04-01
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| Series: | Molecules |
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| Online Access: | https://www.mdpi.com/1420-3049/28/9/3711 |
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| author | Simona Sedláčková Martin Hubálek Vladimír Vrkoslav Miroslava Blechová Petr Kozlík Josef Cvačka |
| author_facet | Simona Sedláčková Martin Hubálek Vladimír Vrkoslav Miroslava Blechová Petr Kozlík Josef Cvačka |
| author_sort | Simona Sedláčková |
| collection | DOAJ |
| description | A typical bottom-up proteomic workflow comprises sample digestion with trypsin, separation of the hydrolysate using reversed-phase HPLC, and detection of peptides via electrospray ionization (ESI) tandem mass spectrometry. Despite the advantages and wide usage of protein identification and quantification, the procedure has limitations. Some domains or parts of the proteins may remain inadequately described due to inefficient detection of certain peptides. This study presents an alternative approach based on sample acetylation and mass spectrometry with atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI). These ionizations allowed for improved detection of acetylated peptides obtained via chymotrypsin or glutamyl peptidase I (Glu-C) digestion. APCI and APPI spectra of acetylated peptides often provided sequence information already at the full scan level, while fragmentation spectra of protonated molecules and sodium adducts were easy to interpret. As demonstrated for bovine serum albumin, acetylation improved proteomic analysis. Compared to ESI, gas-phase ionizations APCI and APPI made it possible to detect more peptides and provide better sequence coverages in most cases. Importantly, APCI and APPI detected many peptides which passed unnoticed in the ESI source. Therefore, analytical methods based on chymotrypsin or Glu-C digestion, acetylation, and APPI or APCI provide data complementary to classical bottom-up proteomics. |
| first_indexed | 2024-03-11T04:12:21Z |
| format | Article |
| id | doaj.art-97444bb57a0941f5bf5e8f339d1380d1 |
| institution | Directory Open Access Journal |
| issn | 1420-3049 |
| language | English |
| last_indexed | 2024-03-11T04:12:21Z |
| publishDate | 2023-04-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Molecules |
| spelling | doaj.art-97444bb57a0941f5bf5e8f339d1380d12023-11-17T23:22:26ZengMDPI AGMolecules1420-30492023-04-01289371110.3390/molecules28093711Positive Effect of Acetylation on Proteomic Analysis Based on Liquid Chromatography with Atmospheric Pressure Chemical Ionization and Photoionization Mass SpectrometrySimona Sedláčková0Martin Hubálek1Vladimír Vrkoslav2Miroslava Blechová3Petr Kozlík4Josef Cvačka5Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Flemingovo Náměstí 542/2, 16000 Prague, Czech RepublicInstitute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Flemingovo Náměstí 542/2, 16000 Prague, Czech RepublicInstitute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Flemingovo Náměstí 542/2, 16000 Prague, Czech RepublicInstitute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Flemingovo Náměstí 542/2, 16000 Prague, Czech RepublicDepartment of Analytical Chemistry, Faculty of Science, Charles University, Hlavova 2030/8, 12800 Prague, Czech RepublicInstitute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Flemingovo Náměstí 542/2, 16000 Prague, Czech RepublicA typical bottom-up proteomic workflow comprises sample digestion with trypsin, separation of the hydrolysate using reversed-phase HPLC, and detection of peptides via electrospray ionization (ESI) tandem mass spectrometry. Despite the advantages and wide usage of protein identification and quantification, the procedure has limitations. Some domains or parts of the proteins may remain inadequately described due to inefficient detection of certain peptides. This study presents an alternative approach based on sample acetylation and mass spectrometry with atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI). These ionizations allowed for improved detection of acetylated peptides obtained via chymotrypsin or glutamyl peptidase I (Glu-C) digestion. APCI and APPI spectra of acetylated peptides often provided sequence information already at the full scan level, while fragmentation spectra of protonated molecules and sodium adducts were easy to interpret. As demonstrated for bovine serum albumin, acetylation improved proteomic analysis. Compared to ESI, gas-phase ionizations APCI and APPI made it possible to detect more peptides and provide better sequence coverages in most cases. Importantly, APCI and APPI detected many peptides which passed unnoticed in the ESI source. Therefore, analytical methods based on chymotrypsin or Glu-C digestion, acetylation, and APPI or APCI provide data complementary to classical bottom-up proteomics.https://www.mdpi.com/1420-3049/28/9/3711acetylationchemical ionizationphotoionizationproteomics |
| spellingShingle | Simona Sedláčková Martin Hubálek Vladimír Vrkoslav Miroslava Blechová Petr Kozlík Josef Cvačka Positive Effect of Acetylation on Proteomic Analysis Based on Liquid Chromatography with Atmospheric Pressure Chemical Ionization and Photoionization Mass Spectrometry Molecules acetylation chemical ionization photoionization proteomics |
| title | Positive Effect of Acetylation on Proteomic Analysis Based on Liquid Chromatography with Atmospheric Pressure Chemical Ionization and Photoionization Mass Spectrometry |
| title_full | Positive Effect of Acetylation on Proteomic Analysis Based on Liquid Chromatography with Atmospheric Pressure Chemical Ionization and Photoionization Mass Spectrometry |
| title_fullStr | Positive Effect of Acetylation on Proteomic Analysis Based on Liquid Chromatography with Atmospheric Pressure Chemical Ionization and Photoionization Mass Spectrometry |
| title_full_unstemmed | Positive Effect of Acetylation on Proteomic Analysis Based on Liquid Chromatography with Atmospheric Pressure Chemical Ionization and Photoionization Mass Spectrometry |
| title_short | Positive Effect of Acetylation on Proteomic Analysis Based on Liquid Chromatography with Atmospheric Pressure Chemical Ionization and Photoionization Mass Spectrometry |
| title_sort | positive effect of acetylation on proteomic analysis based on liquid chromatography with atmospheric pressure chemical ionization and photoionization mass spectrometry |
| topic | acetylation chemical ionization photoionization proteomics |
| url | https://www.mdpi.com/1420-3049/28/9/3711 |
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