TagGraph reveals vast protein modification landscapes from large tandem mass spectrometry datasets.

Although mass spectrometry is well suited to identifying thousands of potential protein post-translational modifications (PTMs), it has historically been biased towards just a few. To measure the entire set of PTMs across diverse proteomes, software must overcome the dual challenges of covering enormous search spaces and distinguishing correct from incorrect spectrum interpretations. Here, we describe TagGraph, a computational tool that overcomes both challenges with an unrestricted string-based search method that is as much as 350-fold faster than existing approaches, and a probabilistic validation model that we optimized for PTM assignments. We applied TagGraph to a published human proteomic dataset of 25 million mass spectra and tripled confident spectrum identifications compared to its original analysis. We identified thousands of modification types on almost 1 million sites in the proteome. We show alternative contexts for highly abundant yet understudied PTMs such as proline hydroxylation, and its unexpected association with cancer mutations. By enabling broad characterization of PTMs, TagGraph informs as to how their functions and regulation intersect.

Antigen presentation profiling reveals recognition of lymphoma immunoglobulin neoantigens.

Cancer somatic mutations can generate neoantigens that distinguish malignant from normal cells. However, the personalized identification and validation of neoantigens remains a major challenge. Here we discover neoantigens in human mantle-cell lymphomas by using an integrated genomic and proteomic strategy that interrogates tumour antigen peptides presented by major histocompatibility complex (MHC) class I and class II molecules. We applied this approach to systematically characterize MHC ligands from 17 patients. Remarkably, all discovered neoantigenic peptides were exclusively derived from the lymphoma immunoglobulin heavy- or light-chain variable regions. Although we identified MHC presentation of private polymorphic germline alleles, no mutated peptides were recovered from non-immunoglobulin somatically mutated genes. Somatic mutations within the immunoglobulin variable region were almost exclusively presented by MHC class II. We isolated circulating CD4+ T cells specific for immunoglobulin-derived neoantigens and found these cells could mediate killing of autologous lymphoma cells. These results demonstrate that an integrative approach combining MHC isolation, peptide identification, and exome sequencing is an effective platform to uncover tumour neoantigens. Application of this strategy to human lymphoma implicates immunoglobulin neoantigens as targets for lymphoma immunotherapy.

One-step immunostrip test for the simultaneous detection of free and total prostate specific antigen in serum.

The development of a one-step lateral flow immunoassay on a strip format for the rapid and reliable simultaneous detection of serum levels of free and total prostate specific antigen (f-PSA and t-PSA) and estimation of f-PSA to t-PSA ratio (f/t-PSA) is reported. The f/t-PSA ratio has shown to be more specific for the correct diagnosis of prostate cancer than t-PSA alone, especially in the so-called diagnostic grey zone of 4-10 microg/l t-PSA. The performance of the system described relied on non-competitive immunoassay protocols. Herein, f-PSA and t-PSA were sandwiched between anti-f-PSA and anti-t-PSA monoclonal antibodies immobilised on the strip and a colloidal gold anti-PSA antibody tracer. In the presence of PSA in the sample, the tracer accumulated on the strip results in the appearance of specific pink colour lines. The colour intensity of these lines was found to be directly proportional to the PSA concentration and a semi-quantitative estimation could be carried out visually. Quantitative analysis was also possible by densitometry. Using PSA standards prepared in female serum, the strip could be calibrated up to a concentration of 60 microg/l for both PSA species, with an assay time of less than 20 min. The estimated detection limit was 1 microg/l in all cases. The immunostrip showed good storage stability for at least 2 months and the reproducibility was always between 12% and 17%. Fifty-one male serum samples were analysed with the strip and results compared with values obtained by two different commercial immunoassays taken as reference methods. The study yielded acceptable correlation and agreement. An estimation of the sensitivity and specificity demonstrated the strip validity as a potential front-line device for the early detection of prostate cancer and differentiation of benign prostatic anomalies. Small plastic cartridges incorporating the immunostrip and a small blister containing washing solution that helps remove unbound species from the strip were envisaged in order to avoid false positive readings and decrease background signals, thereby leading to better sensitivity and detection limits.

Disposable noncompetitive immunosensor for free and total prostate-specific antigen based on capacitance measurement.

This work reports on the successful integration of a one-step lateral flow immunoassay format and impedance detection of the specific affinity event using an electrochemical transducer coated with a pH-sensitive polymer layer. This approach was particularly applied to the development of a rapid single-use immunosensor for the sensitive detection of free and total prostate-specific antigen (f-PSA, t-PSA) tumor marker. Strips of nitrocellulose membrane were coated with appropriate antibodies to f-PSA and t-PSA and used as solid supports for the performance of noncompetitive immunoassays where PSA was allowed to react with both immobilized anti-PSA antibody and anti-PSA urease enzyme conjugate for less than 1 min. An additional piece within the device consisting of a storage blister filled with a urea solution allowed the rapid washing of unbound species from the membrane strips and simultaneous urea hydrolysis catalyzed by the bound urease conjugate in an automatic fashion. The hydrolysis of urea increased the pH of the reaction media, which in turn induced a breakdown of the polymer layer on the transducer and a consequent measurable change in capacitance of the system. This was easily recorded at a given frequency over a 30-min period. Overall, we describe a one-step immunosensor prototype that exhibits enough sensitivity to detect both forms of PSA at concentration levels down to 3 ng/mL. With the possibility of being portable and considering its ease of use, robustness, and simplicity, this device has great potential as a tool for the screening and early detection of prostate cancer.

Disposable tyrosinase-peroxidase bi-enzyme sensor for amperometric detection of phenols.

A new disposable amperometric bi-enzyme sensor system for detecting phenols has been developed. The phenol sensor developed uses horseradish peroxidase modified screen-printed carbon electrodes (HRP-SPCEs) coupled with immobilized tyrosinase prepared using poly(carbamoylsulfonate) (PCS) hydrogels or a poly(vinyl alcohol) bearing styrylpyridinium groups (PVA-SbQ) matrix. Optimization of the experimental parameters has been performed with regard to buffer composition, pH, operating potential and storage stability. A co-operative reaction involving tyrosinase and HRP occurs at a potential of -50 mV versus Ag/AgCl without the requirement for addition of extraneous H(2)O(2), thus, resulting in a very simple and efficient system. Comparison of the electrode responses with the 4-aminoantipyrine standard method for phenol sample analysis indicated the feasibility of the disposable sensor system for sensitive "in-field" determination of phenols. The most sensitive system was the tyrosinase immobilized HRP-SPCE using PCS, which displayed detection limits for phenolic compounds in the lower nanomolar range e.g. 2.5 nM phenol, 10 nM catechol and 5 nM p-cresol.