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.

• Klíčová slova
• acetylation, chemical ionization, photoionization, proteomics,
• MeSH
• acetylace MeSH
• atmosférický tlak MeSH
• chymotrypsin * MeSH
• hmotnostní spektrometrie s elektrosprejovou ionizací metody   MeSH
• peptidy MeSH
• proteomika * MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• chymotrypsin * MeSH
• peptidy MeSH

Electrospray ionization of methanolic solutions of p-hydroxyphenacyl derivatives HO-C(6)H(4)-C(O)-CH(2)-X (X = leaving group) provides abundant signals for the deprotonated species which are assigned to the corresponding phenolate anions (-)O-C(6)H(4)-C(O)-CH(2)-X. Upon collisional activation in the gas phase, these anions inter alia undergo loss of a neutral "C(8)H(6)O(2)" species concomitant with formation of the corresponding anions X(-). The energies required for the loss of the neutral roughly correlate with the gas phase acidities of the conjugate acids (HX). Extensive theoretical studies performed for X = CF(3)COO in order to reveal the energetically most favorable pathway for the formation of neutral "C(8)H(6)O(2)" suggest three different routes of similar energy demands, involving a spirocyclopropanone, epoxide formation, and a diradical, respectively.

• MeSH
• acetofenony chemie   MeSH
• anionty chemie   MeSH
• cyklopropany chemie   MeSH
• epoxidové sloučeniny chemie   MeSH
• hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
• molekulární struktura MeSH
• roztoky chemie   MeSH
• spirosloučeniny chemie   MeSH
• teoretické modely MeSH
• vzácné plyny chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• acetofenony MeSH
• anionty MeSH
• cyklopropany MeSH
• epoxidové sloučeniny MeSH
• roztoky MeSH
• spirosloučeniny MeSH
• vzácné plyny MeSH