Utilizing Precursor Ion Connectivity of Different Charge States to Improve Peptide and Protein Identification in MS/MS Analysis.

Tandem mass spectrometry (MS/MS) has become a key method for the structural analysis of biomolecules such as peptides and proteins. A pervasive problem in MS/MS analyses, especially for top-down proteomics, is the occurrence of chimeric spectra, when two or more precursor ions are co-isolated and fragmented, thus leading to complex MS/MS spectra that are populated with fragment ions originating from different precursor ions. This type of convoluted data typically results in low sequence database search scores due to the vast number of mixed-source fragment ions, of which only a fraction originates from a specific precursor ion. Herein, we present a novel workflow that deconvolutes the data of chimeric MS/MS spectra, improving the protein search scores and sequence coverages in database searching and thus providing a more confident peptide and protein identification. Previously misidentified proteins or proteins with insignificant search scores can be correctly and significantly identified following the presented data acquisition and analysis workflow with search scores increasing by a factor of 3-4 for smaller precursor ions (peptides) and >6 for larger precursor ions such as intact ubiquitin and cytochrome C.

Liquid Atmospheric Pressure Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Using a Commercial Ion Source and Orbitrap Mass Analyzer.

A liquid atmospheric pressure-matrix-assisted laser desorption/ionization (LAP-MALDI) method has been developed and applied to a commercial AP-MALDI source on a hybrid orbitrap mass analyzer. It is shown that electrospray ionization (ESI)-like mass spectra of a range of peptides and proteins can be acquired by LAP-MALDI mass spectrometry (MS) as previously demonstrated on a homemade LAP-MALDI-Q-TOF setup but without the need of any modification to the commercially available MS equipment used. Multiply charged peptide ions were recorded with a resolution of around 100,000 and a mass accuracy of less than 5 ppm. The higher resolution and mass accuracy of the orbitrap analyzer compared with previously employed Q-TOF instrumentation provided high confidence in bacterial proteoform and species identification by top-down protein analysis.