Applications of Mass Spectrometry Imaging to Cancer.

Pathologists play an essential role in the diagnosis and prognosis of benign and cancerous tumors. Clinicians provide tissue samples, for example, from a biopsy, which are then processed and thin sections are placed onto glass slides, followed by staining of the tissue with visible dyes. Upon processing and microscopic examination, a pathology report is provided, which relies on the pathologist's interpretation of the phenotypical presentation of the tissue. Targeted analysis of single proteins provide further insight and together with clinical data these results influence clinical decision making. Recent developments in mass spectrometry facilitate the collection of molecular information about such tissue specimens. These relatively new techniques generate label-free mass spectra across tissue sections providing nonbiased, nontargeted molecular information. At each pixel with spatial coordinates (x/y) a mass spectrum is acquired. The acquired mass spectrums can be visualized as intensity maps displaying the distribution of single m/z values of interest. Based on the sample preparation, proteins, peptides, lipids, small molecules, or glycans can be analyzed. The generated intensity maps/images allow new insights into tumor tissues. The technique has the ability to detect and characterize tumor cells and their environment in a spatial context and combined with histological staining, can be used to aid pathologists and clinicians in the diagnosis and management of cancer. Moreover, such data may help classify patients to aid therapy decisions and predict outcomes. The novel complementary mass spectrometry-based methods described in this chapter will contribute to the transformation of pathology services around the world.

An immunodominant La/SSB autoantibody proteome derives from public clonotypes.

The La/SSB autoantigen is a major target of long-term humoral autoimmunity in primary Sjögren's Syndrome (SS) and systemic lupus erythematosus. A majority of patients with linked anti-Ro60/Ro52/La responses target an NH2-terminal epitope designated LaA that is expressed on Ro/La ribonucleoprotein complexes and the surface membrane of apoptotic cells. In this study, we used high-resolution Orbitrap mass spectrometry to determine the clonality, isotype and V-region sequences of LaA-specific autoantibodies in seven patients with primary SS. Anti-LaA immunoglobulin (Ig)Gs purified from polyclonal sera by epitope-specific affinity chromatography were analysed by combined database and de-novo mass spectrometric sequencing. Autoantibody responses comprised two heavily mutated IgG1 kappa-restricted monoclonal species that were shared (public) across unrelated patients; one clonotype was specified by an IGHV3-30 heavy chain paired with IGKV3-15 light chain and the second by an IGHV3-43/IGKV3-20 pairing. Shared amino acid replacement mutations were also seen within heavy and light chain complementarity-determining regions, consistent with a common breach of B cell tolerance followed by antigen-driven clonal selection. The discovery of public clonotypic autoantibodies directed against an immunodominant epitope on La, taken together with recent findings for the linked Ro52 and Ro60 autoantigens, supports a model of systemic autoimmunity in which humoral responses against protein-RNA complexes are mediated by public sets of autoreactive B cell clonotypes.