Peroxynitrite in the tumor microenvironment changes the profile of antigens allowing escape from cancer immunotherapy.

Cancer immunotherapy often depends on recognition of peptide epitopes by cytotoxic T lymphocytes (CTLs). The tumor microenvironment (TME) is enriched for peroxynitrite (PNT), a potent oxidant produced by infiltrating myeloid cells and some tumor cells. We demonstrate that PNT alters the profile of MHC class I bound peptides presented on tumor cells. Only CTLs specific for PNT-resistant peptides have a strong antitumor effect in vivo, whereas CTLs specific for PNT-sensitive peptides are not effective. Therapeutic targeting of PNT in mice reduces resistance of tumor cells to CTLs. Melanoma patients with low PNT activity in their tumors demonstrate a better clinical response to immunotherapy than patients with high PNT activity. Our data suggest that intratumoral PNT activity should be considered for the design of neoantigen-based therapy and also may be an important immunotherapeutic target.

Proteome Analysis Using Gel-LC-MS/MS.

This article describes processing of protein samples using 1D SDS gels prior to protease digestion for proteomics workflows that subsequently utilize reversed-phase nanocapillary ultra-high-pressure liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS). The resulting LC-MS/MS data are used to identify peptides and thereby infer proteins present in samples ranging from simple mixtures to very complex proteomes. Bottom-up proteome studies usually involve quantitative comparisons across several or many samples. For either situation, 1D SDS gels represent a simple, widely available technique that can be used to either fractionate complex proteomes or rapidly clean up low microgram samples with minimal losses. After gel separation and staining/destaining, appropriate gel slices are excised, and in-gel reduction, alkylation, and protease digestion are performed. Digests are then processed for LC-MS/MS analysis. Protocols are described for either sample fractionation with high-throughput processing of many samples or simple cleanup without fractionation. An optional strategy is to conduct in-solution reduction and alkylation prior to running gels, which is advantageous when a large number of samples will be separated into large numbers of fractions. Optimization of trypsin digestion parameters and comparison to in-solution protease digestion are also described. © 2019 by John Wiley & Sons, Inc.

Full-Length Anion Exchanger 1 Structure and Interactions with Ankyrin-1 Determined by Zero Length Crosslinking of Erythrocyte Membranes.

Anion exchanger 1 (AE1) is a critical transporter and the primary structural scaffold for large macromolecular complexes responsible for erythrocyte membrane flexibility and integrity. We used zero-length crosslinking and mass spectrometry to probe AE1 structures and interactions in intact erythrocyte membranes. An experimentally verified full-length model of AE1 dimers was developed by combining crosslink-defined distance constraints with homology modeling. Previously unresolved cytoplasmic loops in the AE1 C-terminal domain are packed at the domain-domain interface on the cytoplasmic face of the membrane where they anchor the N-terminal domain's location and prevent it from occluding the ion channel. Crosslinks between AE1 dimers and ankyrin-1 indicate the likely topology for AE1 tetramers and suggest that ankyrin-1 wraps around AE1 tetramers, which may stabilize this oligomer state. This interaction and interactions of AE1 with other major erythrocyte membrane proteins show that protein-protein contacts are often substantially more extensive than previously reported.

Enhanced analysis of human breast cancer proteomes using micro-scale solution isoelectrofocusing combined with high resolution 1-D and 2-D gels.

Current methods for quantitatively comparing proteomes (protein profiling) have inadequate resolution and dynamic range for complex proteomes such as those from mammalian cells or tissues. More extensive profiling of complex proteomes would be obtained if the proteomes could be reproducibly divided into a moderate number of well-separated pools. But the utility of any prefractionation is dependent upon the resolution obtained because extensive cross contamination of many proteins among different pools would make quantitative comparisons impractical. The current study used a recently developed microscale solution isoelectrofocusing (musol-IEF) method to separate human breast cancer cell extracts into seven well-resolved pools. High resolution fractionation could be achieved in a series of small volume tandem chambers separated by thin acrylamide partitions containing covalently bound immobilines that establish discrete pH zones to separate proteins based upon their pIs. In contrast to analytical 2-D gels, this prefractionation method was capable of separating very large proteins (up to about 500 kDa) that could be subsequently profiled and quantitated using large-pore 1-D SDS gels. The pH 4.5-6.5 region was divided into four 0.5 pH unit ranges because this region had the greatest number of proteins. By using very narrow pH range fractions, sample amounts applied to narrow pH range 2-D gels could be increased to detect lower abundance proteins. Although 1.0 pH range 2-D gels were used in these experiments, further protein resolution should be feasible by using 2-D gels with pH ranges that are only slightly wider than the pH ranges of the musol-IEF fractions. By combining musol-IEF prefractionation with subsequent large pore 1-D SDS-PAGE (>100 kDa) and narrow range 2-D gels (<100 kDa), large proteins can be reliably quantitated, many more proteins can be resolved, and lower abundance proteins can be detected.