Utility of Atmospheric-Pressure Chemical Ionization and Photoionization Mass Spectrometry in Bottom-Up Proteomics

Abstract

In a typical bottom-up proteomics workflow, proteins are enzymatically cleaved, and the resulting peptides are analyzed by HPLC with electrospray ionization (ESI) tandem mass spectrometry. This approach is practical and widely applied. It has, however, limitations mostly related to less efficient or even inefficient ionization of some peptides in ESI sources. Gas-phase ionization methods like atmospheric-pressure chemical ionization (APCI) or atmospheric-pressure photoionization (APPI) offer alternative ways of detecting various analytes. This work is a systematic study of the ionization efficiencies of peptides in ESI, APCI, and APPI and the applicability of the mentioned ionizations in proteomics. A set of peptide standards and bovine serum albumin digests were examined using a high-resolution mass spectrometer coupled to an ultra HPLC system. Since the ionization efficiency in APCI and APPI depends strongly on experimental conditions, the ion source settings and mobile phase compositions were optimized for each ionization technique. As expected, tryptic peptides were best detected using ESI. The numbers of chymotrypsin peptides successfully detected by ESI, APPI, and APCI were comparable. In the case of Glu-C digest, APPI detected the highest number of peptides. The results suggest that gas-phase ionization techniques, particularly APPI, are an interesting alternative for detecting peptides and delivering complementary data in proteomics.

Positive Effect of Acetylation on Proteomic Analysis Based on Liquid Chromatography with Atmospheric Pressure Chemical Ionization and Photoionization Mass Spectrometry

“…APPI proved effective for detecting hydrophobic peptides with a higher molecular weight [35,36], as well as PTMs such as palmitoylation [37,38]. In our recent study, both gas-phase ionization techniques efficiently detected peptides from Glu-C and chymotrypsin digests [39].…”

“…The sodiated peptide precursors also yielded a ions, likely due to the loss of CO from b ions [72]. Regarding eliminating small neutral molecules from the peptide precursors, NH 3 and H 2 O are typical losses for non-acetylated peptides [39]. Neutral loss of NH 3 was rarely detected for acetylated peptides; instead, elimination of H 2 O, CO, and HCHO occurred.…”

“…In addition to providing separation, the mobile phase must also effectively support the ionization of the analytes. As regards gas-phase ionization methods, i.e., APCI and APPI, we have previously shown that ionization of peptides is most efficient when no buffer is present and the mobile phase is neutral or basic [39]. Here we tested the signals of acetylated SLGE, SLGK, VASLF, AWSVAF, AWSVAE, VLASSAE, and QTALVELLF in acetonitrile and methanol combined with water or aqueous ammonium hydroxide (pH 9).…”

“…BSA digests were used to test the applicability of acetylation in bottom-up proteomic workflows based on APCI and APPI. The digests were prepared using chymotrypsin or Glu-C, which were shown to provide peptides with better properties than trypsin for gas-phase ionizations [39].…”

Data‐Driven Compound Identification in Atmospheric Mass Spectrometry

“…The experimentally resolvable fraction of compounds, in terms of their volatility, has expanded steadily, as techniques have improved. [31,40] For example, large biomolecules have been detected using several spray ionization sources (e.g., electrospray ionization [ESI] [41,42] and atmospheric pressure photoionization [APPI]), [43][44][45] and surface-bound species by desorption techniques such as matrix-assisted laser desorption ionization (MALDI). [31,46] Particulate bound targets, the constituents of nanoparticles, can be detected through direct aerosol sampling by, for example, using an aerodynamic lens with subsequent flash vaporization and EI ionization in aerosol mass spectrometry (AMS), [47] or by collecting the particles onto a filter (or wire) with subsequent rapid thermal desorption vaporization of the condensed-phase constituents.…”