Aliphatic hydrocarbons (HCs) are usually analyzed by gas chromatography (GC) or matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. However, analyzing long-chain HCs by GC is difficult because of their low volatility and the risk of decomposition at high temperatures. MALDI cannot distinguish between isomeric HCs. An alternative approach based on silver ion high-performance liquid chromatography (Ag-HPLC) is shown here. The separation of HC standards and cuticular HCs was accomplished using two ChromSpher Lipids columns connected in series. A gradient elution of the analytes was optimized using mobile phases prepared from hexane (or isooctane) and acetonitrile, 2-propanol, or toluene. HCs were detected by atmospheric pressure chemical ionization mass spectrometry (APCI-MS). Good separation of the analytes according to the number of double bonds, cis/trans geometry, and position of double bonds was achieved. The retention times increased with the number of double bonds, and trans isomers eluted ahead of cis isomers. The mobile phase significantly affected the mass spectra of HCs. Depending on the mobile phase composition, deprotonated molecules, molecular ions, protonated molecules, and various solvent-related adducts of HCs were observed. The optimized Ag-HPLC/APCI-MS was applied for characterizing cuticular HCs from a flesh fly, Neobellieria bullata, and cockroach, Periplaneta americana. The method made it possible to detect a significantly higher number of HCs than previously reported for GC or MALDI-MS. Unsaturated HCs were frequently detected as isomers differing by double-bond position(s). Minor HCs with trans double bonds were found beside the prevailing cis isomers. Ag-HPLC/APCI-MS has great potential to become a new tool in chemical ecology for studying cuticular HCs.

• Klíčová slova
• Neobellieria bullata, Periplaneta americana, double bonds, hydrocarbons, mass spectrometry, semiochemicals,
• MeSH
• atmosférický tlak MeSH
• plynová chromatografie s hmotnostně spektrometrickou detekcí MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• stříbro * chemie   MeSH
• uhlovodíky * MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• stříbro * MeSH
• uhlovodíky * MeSH

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 may exhibit inadequate ionization efficiency in some applications. In such cases, atmospheric-pressure chemical ionization (APCI) and photoionization (APPI) can be used. Despite a wide application potential, no APCI and APPI sources dedicated to very low sample flow rates exist on the market. Since the ion source performance depends on the transfer of analytes from the liquid to the gas phase, a nebulizer is a critical component of an ion source. Here, we report on the nebulizer with a gas dynamic virtual nozzle (GDVN) and its applicability in APCI at microliter-per-minute flow rates. Nebulizers differing by geometrical parameters were fabricated and characterized regarding the jet breakup regime, droplet size, droplet velocity, and spray angle for liquid flow rates of 0.75-15.0 μL/min. A micro-APCI source with the GDVN nebulizer behaved as a mass-flow-sensitive detector and provided stable and intense analyte signals. Compared to a classical APCI source, an order of magnitude lower detection limit for verapamil was achieved. Mass spectra recorded with the nebulizer in dripping and jetting modes were almost identical and did not differ from normal APCI spectra. Clogging never occurred during the experiments, indicating the high robustness of the nebulizer. Low-flow-rate APCI and APPI sources with a GDVN sprayer promise new applications for low- and medium-polar analytes.

• Publikační typ
• časopisecké články MeSH