Tandem mass spectrometry of homologous 3-hydroxyfurazan and nitrile amino acids: Analysis of cooperative interactions and fragmentation processes.

The assignment of structure by tandem mass spectrometry (MS/MS) relies on the interpretation of the fragmentation behavior of gas-phase ions. Mass spectra were acquired for a series of heterocyclic mimetics of acidic amino acids and a related series of nitrile amino acids. All amino acids were readily protonated or deprotonated by electrospray ionization (ESI), and distinctive fragmentation processes were observed when the ions were subjected to collision-induced dissociation (CID).

Phenyl group participation in rearrangements during collision-induced dissociation of deprotonated phenoxyacetic acid.

Rationale: The identification of trace constituents in biological and environmental samples is frequently based on the fragmentation patterns resulting from the collision-induced dissociation (CID) of gas-phase ions. Credible mechanistic characterization of fragmentation processes, including rearrangements, is required to make reliable assignments for structures of precursor and product ions.

Methods: Mass spectra were collected using both ion trap and triple quadrupole mass spectrometers operating in the negative ion mode.

A two-stage spin cartridge for integrated protein precipitation, digestion and SDS removal in a comparative bottom-up proteomics workflow.

Unlabelled: Protein precipitation with organic solvent is an effective means of depleting contaminants such as sodium dodecyl sulfate (SDS), while maintaining high analyte recovery. Here, we report the use of a disposable two-stage spin cartridge to facilitate isolation of the precipitated protein, with subsequent enzyme digestion and peptide cleanup in the cartridge. An upper filtration cartridge retains over 95% of the protein (10 μg BSA), with 99.

Maximizing recovery of water-soluble proteins through acetone precipitation.

Solvent precipitation is commonly used to purify protein samples, as seen with the removal of sodium dodecyl sulfate through acetone precipitation. However, in its current practice, protein loss is believed to be an inevitable consequence of acetone precipitation. We herein provide an in depth characterization of protein recovery through acetone precipitation.

Critical assessment of the spectroscopic activity assay for monitoring trypsin activity in organic-aqueous solvent.

Solvent-assisted protein digestion involves enzymatic hydrolysis in mixed aqueous-organic solvents. With trypsin, acetonitrile is the modifying solvent of choice, recommended at concentrations from 10 to 80% to improve protein sequence coverage in mass spectrometry. Spectroscopic activity assays employing substrate mimics such as N-benzoyl arginine ethyl ester (BAEE) appear to show a relative enhancement of trypsin activity in mixed solvent systems.

Perfluorooctanoic acid and ammonium perfluorooctanoate: volatile surfactants for proteome analysis?

Rationale: Fluorinated surfactants are being explored as mass spectrometry (MS)-friendly alternatives to sodium dodecyl sulfate (SDS) for proteome analysis. Previous work demonstrates perfluorooctanoic acid (PFOA) to be compatible with electrospray ionization (ESI)-MS. The high volatility of PFOA provides an intrinsic approach to potentially eliminate the surfactant during ESI, or alternatively through solvent evaporation prior to MS.

Implications of partial tryptic digestion in organic-aqueous solvent systems for bottom-up proteome analysis.

For bottom-up MS, the digestion step is critical and is typically performed with trypsin. Solvent-assisted digestion in 80% acetonitrile has previously been shown to improve protein sequence coverage at shorter digestion times. This has been attributed to enhanced enzyme digestion efficiency in this solvent.