Characterization of drug-product-related impurities and variants of a therapeutic monoclonal antibody by higher energy C-trap dissociation mass spectrometry.

Mass spectrometry (MS) characterization of recombinant monoclonal antibody (mAb) drugs and their degraded and/or post-translationally modified counterparts, drug-product-related impurities and variants, is critical for successful development of biotherapeutics. Specifically in this study, drug-product-related impurities of an anti-Clostridium difficile IgG1 mAb drug substance were profiled by cation-exchange liquid chromatography (CEX) followed by the CEX peaks being fraction-collected for MS characterization. A reversed-phase liquid chromatography/mass spectrometry (LC/MS) methodology was developed on a Thermo Q-Exactive orbitrap mass spectrometer for (1) accurate mass measurements of the mAb, its CEX fractionated impurities, and their respective heavy chains and light chains and (2) middle-down LC/MS/MS of the light chains and the heavy chains using higher energy C-trap dissociation (HCD).

Deconvolution and database search of complex tandem mass spectra of intact proteins: a combinatorial approach.

Top-down proteomics studies intact proteins, enabling new opportunities for analyzing post-translational modifications. Because tandem mass spectra of intact proteins are very complex, spectral deconvolution (grouping peaks into isotopomer envelopes) is a key initial stage for their interpretation. In such spectra, isotopomer envelopes of different protein fragments span overlapping regions on the m/z axis and even share spectral peaks.

Phyloproteomic classification of unsequenced organisms by top-down identification of bacterial proteins using capLC-MS/MS on an Orbitrap.

Currently, most MS-based proteomic studies of bacteria and archea match experimental data to known amino acid sequences from the target organism. Top-down studies use a protein's molecular weight along with data gathered from MS/MS experiments to identify proteins by database matching. For Erwinia herbicola and Enterobacter cloacae, studied here, the necessary protein sequences are not available in protein sequence repositories.

High-throughput middle-down analysis using an orbitrap.

This report demonstrates the application of a capillary LC-LTQ-orbitrap system to provide automated middle-down analysis of proteolytic peptides in the mass range 3000 to 10,000 Da. The novel workflow combines an underutilized method in the orbitrap-high resolution, mass-accurate product ion measurements-with software tailored to search such data (ProSightPC 2.0) and an Asp-selective chemical cleavage approach that generates peptides across an extended mass range.

Top-down identification of protein biomarkers in bacteria with unsequenced genomes.

MALDI mass spectrometry-based systems for rapid characterization of microorganisms in biodefense or medical diagnostics usually detect intact proteins in the 5000-20,000 Da range. To evaluate the reliability of species discrimination, and also for forensic applications, it is important that these biomarker proteins be identified. In the present study we apply high resolution tandem mass analysis on an Orbitrap and top-down bioinformatics to identify major biomarker proteins observed in MALDI spectra of intact bacteria for which little genomic or protein sequence information is available.

Evaluation of microwave-accelerated residue-specific acid cleavage for proteomic applications.

Microwave-accelerated proteolysis using acetic acid has been shown to occur specifically on either or both sides of aspartic acid residues. This chemical cleavage has been applied to ovalbumin and several model peptides to test the effect on some of the more common post-translational modifications. No oxidation of methionine or cysteine was observed; however, hydrolysis of phosphate groups proceeds at a detectable rate.