RESUMEN
Negative ion mode nanoelectrospray ionization (nESI) is often utilized to analyze acidic compounds, from small molecules to proteins, with mass spectrometry (MS). Under high aqueous solvent conditions, corona discharge is commonly observed at emitter tips, resulting in low ion abundances and reduced nESI needle lifetimes. We have successfully reduced corona discharge in negative ion mode by trace addition of trifluoroethanol (TFE) to aqueous samples. The addition of as little as 0.2% TFE increases aqueous spray stability not only in nESI direct infusion, but also in nanoflow liquid chromatography (nLC)/MS experiments. Negative ion mode spray stability with 0.2% TFE is approximately 6× higher than for strictly aqueous samples. Upon addition of 0.2% TFE to the mobile phase of nLC/MS experiments, tryptic peptide identifications increased from 93 to 111 peptides, resulting in an average protein sequence coverage increase of 18%.
Asunto(s)
Nanotecnología , Espectrometría de Masa por Ionización de Electrospray/métodos , Trifluoroetanol/química , Cromatografía Líquida de Alta Presión , Iones/química , Péptidos/análisis , Agua/químicaRESUMEN
Protein S-sulfinylation (R-SO2-) and S-sulfonylation (R-SO3-) are irreversible oxidative post-translational modifications of cysteine residues. Greater than 5% of cysteines are reported to occupy these higher oxidation states, which effectively inactivate the corresponding thiols and alter the electronic and physical properties of modified proteins. Such higher oxidation states are reached after excessive exposure to cellular oxidants, and accumulate across different disease states. Despite widespread and functionally relevant cysteine oxidation across the proteome, there are currently no robust methods to profile higher order cysteine oxidation. Traditional data-dependent liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods generally miss low-occupancy modifications in complex analyses. Here, we present a data-independent acquisition (DIA) LC/MS-based approach, leveraging the high IR absorbance of sulfoxides at 10.6 µm, for selective dissociation and discovery of S-sulfonated peptides. Across peptide standards and protein digests, we demonstrate selective infrared multiphoton dissociation (IRMPD) of S-sulfonated peptides in the background of unmodified peptides. This selective DIA IRMPD LC/MS-based approach allows identification and annotation of S-sulfonated peptides across complex mixtures while providing sufficient sequence information to localize the modification site.