MALDI mass spectrometry imaging is able to simultaneously determine the spatial distribution of hundreds of molecules directly from tissue sections, without labeling and without prior knowledge. Ultra-high mass resolution measurements based on Fourier-transform mass spectrometry have been utilized to resolve isobaric lipids, metabolites and tryptic peptides. Here we demonstrate the potential of 15T MALDI-FTICR MSI for molecular pathology in a mouse model of high-grade glioma. The high mass accuracy and resolving power of high field FTICR MSI enabled tumor specific proteoforms, and tumor-specific proteins with overlapping and isobaric isotopic distributions to be clearly resolved. The protein ions detected by MALDI MSI were assigned to proteins identified by region-specific microproteomics (0.8 mm2 regions isolated using laser capture microdissection) on the basis of exact mass and isotopic distribution. These label free quantitative experiments also confirmed the protein expression changes observed by MALDI MSI and revealed changes in key metabolic proteins, which were supported by in-situ metabolite MALDI MSI.

The Consortium for Top-Down Proteomics (www.topdownproteomics.org) launched the present study to assess the current state of top-down mass spectrometry (TD MS) and middle-down mass spectrometry (MD MS) for characterizing monoclonal antibody (mAb) primary structures, including their modifications. To meet the needs of the rapidly growing therapeutic antibody market, it is important to develop analytical strategies to characterize the heterogeneity of a therapeutic product's primary structure accurately and reproducibly. The major objective of the present study is to determine whether current TD/MD MS technologies and protocols can add value to the more commonly employed bottom-up (BU) approaches with regard to confirming protein integrity, sequencing variable domains, avoiding artifacts, and revealing modifications and their locations. We also aim to gather information

The emergence of complex protein therapeutics in general and monoclonal antibodies (mAbs) in particular have stimulated analytical chemists to develop new methods and strategies for their structural characterization. Mass spectrometry plays a key role in providing information on the primary amino acid sequence, post-translational modifications, and other structure characteristics that must be monitored during the manufacturing process and subsequent quality control assessment. In this study, we present a novel method that allows structural characterization of mAbs based on MALDI in-source decay (ISD) fragmentation, coupled with Fourier transform ion cyclotron resonance (FT-ICR) MS. The method benefits from higher resolution of absorption mode FT mass spectra, compared to magnitude mode, which enables simultaneous identification of ISD fragments from both the heavy and light chains with a higher confidence in a wide mass range up to m/z 13 500. This method was applied to two standard mAbs, namely NIST mAb and trastuzumab, in preparation for method application in an interlaboratory study on mAbs structural analysis coordinated by the Consortium for Top-Down Proteomics. Extensive sequence coverage was obtained from the middle-down analysis (IdeS- and GingisKHAN-digested mAbs) that complemented the top-down analysis of intact mAbs. In addition, MALDI FT-ICR MS of IdeS-digested mAbs allowed isotopic-level profiling of proteoforms with regard to heavy chain N-glycosylation.

High-throughput mass spectrometry (MS) glycomics is an emerging field driven by technological advancements including sample preparation and data processing. Previously, we reported an automated protocol for the analysis of N-glycans released from plasma proteins that included sialic acid derivatization with linkage-specificity, namely, ethylation of α2,6-linked sialic acid residues and lactone formation of α2,3-linked sialic acids. In the current study, each step in this protocol was further optimized. Method improvements included minimizing the extent of side-reaction during derivatization, an adjusted glycan purification strategy and mass analysis of the released N-glycans by ultrahigh resolution matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance MS. The latter resolved peak overlap and simplified spectral alignment due to high mass measurement precision. Moreover, this resulted in more confident glycan assignments and improved signal-to-noise for low-abundant species. The performance of the protocol renders high-throughput applications feasible in the exciting field of clinical glycomics.