Cell-specific Bioorthogonal Tagging of Glycoproteins

Altered glycoprotein expression is an undisputed corollary of cancer development. Understanding these alterations is paramount but hampered by limitations underlying cellular model systems. For instance, the intricate interactions between tumour and host cannot be adequately recapitulated in monoculture of tumour-derived cell lines. More complex co-culture models usually rely on sorting procedures for proteome analyses and rarely capture the details of protein glycosylation. Here, we report a strategy termed Bio-Orthogonal Cell line-specific Tagging of Glycoproteins (BOCTAG). Cells are equipped by transfection with an artificial biosynthetic pathway that transforms bioorthogonally tagged sugars into the corresponding nucleotide-sugars. Only transfected cells incorporate bioorthogonal tags into glycoproteins in the presence of non-transfected cells. We employ BOCTAG as an imaging technique and to annotate cell-specific glycosylation sites in mass spectrometry-glycoproteomics. We demonstrate application in co-culture and mouse models, allowing for profiling of the glycoproteome as an important modulator of cellular function

Cloud Computing Based Immunopeptidomics Utilizing Community Curated Variant Libraries Simplifies and Improves Neo-Antigen Discovery in Metastatic Melanoma

Unique peptide neo-antigens presented on the cell surface are attractive targets for researchers in nearly all areas of personalized medicine. Cells presenting peptides with mutated or other non-canonical sequences can be utilized for both targeted therapies and diagnostics. Today’s state-of-the-art pipelines utilize complementary proteogenomic approaches where RNA or ribosomal sequencing data helps to create libraries from which tandem mass spectrometry data can be compared. In this study, we present an alternative approach whereby cloud computing is utilized to power neo-antigen searches against community curated databases containing more than 7 million human sequence variants. Using these expansive databases of high-quality sequences as a reference, we reanalyze the original data from two previously reported studies to identify neo-antigen targets in metastatic melanoma. Using our approach, we identify 79 percent of the non-canonical peptides reported by previous genomic analyses of these files. Furthermore, we report 18-fold more non-canonical peptides than previously reported. The novel neo-antigens we report herein can be corroborated by secondary analyses such as high predicted binding affinity, when analyzed by well-established tools such as NetMHC. Finally, we report 738 non-canonical peptides shared by at least five patient samples, and 3258 shared across the two studies. This illustrates the depth of data that is present, but typically missed by lower statistical power proteogenomic approaches. This large list of shared peptides across the two studies, their annotation, non-canonical origin, as well as MS/MS spectra from the two studies are made available on a web portal for community analysis

Acetonitrile Ion Suppression in Atmospheric Pressure Ionization Mass Spectrometry

Efforts to analyze trace levels of cyclic peroxides by liquid chromatography/mass spectrometry gave evidence that acetonitrile suppressed ion formation. Further investigations extended this discovery to ketones, linear peroxides, esters, and possibly many other types of compounds, including triazole and menadione. Direct ionization suppression caused by acetonitrile was observed for multiple adduct types in both electrospray ionization and atmospheric pressure chemical ionization. The addition of only 2% acetonitrile significantly decreased the sensitivity of analyte response. Efforts to identify the mechanism were made using various nitriles. The ion suppression was reduced by substitution of an acetonitrile hydrogen with an electron-withdrawing group, but was exacerbated by electron-donating or steric groups adjacent to the nitrile. Although current theory does not explain this phenomenon, we propose that polar interactions between the various functionalities and the nitrile may be forming neutral aggregates that manifest as ionization suppression. [Figure not available: see fulltext.

MHC-restricted phosphopeptide antigens: preclinical validation and first-in-humans clinical trial in participants with high-risk melanoma

Background Phosphorylated peptides presented by MHC molecules represent a new class of neoantigens expressed on cancer cells and recognized by CD8 T-cells. These peptides are promising targets for cancer immunotherapy. Previous work identified an HLA-A*0201-restricted phosphopeptide from insulin receptor substrate 2 (pIRS2) as one such target. The purpose of this study was to characterize a second phosphopeptide, from breast cancer antiestrogen resistance 3 (BCAR3), and to evaluate safety and immunogenicity of a novel immunotherapic vaccine comprising either or both of these phosphorylated peptides.Methods Phosphorylated BCAR3 protein was evaluated in melanoma and breast cancer cell lines by Western blot, and recognition by T-cells specific for HLA-A*0201-restricted phosphorylated BCAR3 peptide (pBCAR3126-134) was determined by 51Cr release assay and intracellular cytokine staining. Human tumor explants were also evaluated by mass spectrometry for presentation of pIRS2 and pBCAR3 peptides. For the clinical trial, participants with resected stage IIA–IV melanoma were vaccinated 6 times over 12 weeks with one or both peptides in incomplete Freund’s adjuvant and Hiltonol (poly-ICLC). Adverse events (AEs) were coded based on National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) V.4.03, with provision for early study termination if dose-limiting toxicity (DLT) rates exceeded 33%. The enrollment target was 12 participants evaluable for immune response to each peptide. T-cell responses were assessed by interferon-γ ELISpot assay.Results pBCAR3 peptides were immunogenic in vivo in mice, and in vitro in normal human donors, and T-cells specific for pBCAR3126-134 controlled outgrowth of a tumor xenograft. The pIRS21097-1105 peptide was identified by mass spectrometry from human hepatocellular carcinoma tumors. In the clinical trial, 15 participants were enrolled. All had grade 1 or 2 treatment-related AEs, but there were no grade 3–4 AEs, DLTs or deaths on study. T-cell responses were induced to the pIRS21097-1105 peptide in 5/12 patients (42%, 90% CI 18% to 68%) and to the pBCAR3126-134 peptide in 2/12 patients (17%, 90% CI 3% to 44%).Conclusion This study supports the safety and immunogenicity of vaccines containing the cancer-associated phosphopeptides pBCAR3126-134 and pIRS21097-1105, and the data support continued development of immune therapy targeting phosphopeptides. Future studies will define ways to further enhance the magnitude and durability of phosphopeptide-specific immune responses.Trial registration number NCT0184614