Enhancing Data Reliability in TOMAHAQ for Large-Scale Protein Quantification.

The analytical scale of most mass-spectrometry-based targeted proteomics assays is usually limited by assay performance and instrument utilization. A recently introduced method, called triggered by offset, multiplexed, accurate mass, high resolution, and absolute quantitation (TOMAHAQ), combines both peptide and sample multiplexing to simultaneously improve analytical scale and quantitative performance. In the present work, critical technical requirements and data analysis considerations for successful implementation of the TOMAHAQ technique based on the study of a total of 185 target peptides across over 200 clinical plasma samples are discussed. Importantly, it is observed that significant interference originate from the TMTzero reporter ion used for the synthetic trigger peptides. This interference is not expected because only TMT10plex reporter ions from the target peptides should be observed under typical TOMAHAQ conditions. In order to unlock the great promise of the technique for high throughput quantification, here a post-acquisition data correction strategy to deconvolute the reporter ion superposition and recover reliable data is proposed.

The ABRF Proteomics Research Group studies: educational exercises for qualitative and quantitative proteomic analyses.

Resource (core) facilities have played an ever-increasing role in furnishing the scientific community with specialized instrumentation and expertise for proteomics experiments in a cost-effective manner. The Proteomics Research Group (PRG) of the Association of Biomolecular Resource Facilities (ABRF) has sponsored a number of research studies designed to enable participants to try new techniques and assess their capabilities relative to other laboratories analyzing the same samples. Presented here are results from three PRG studies representing different samples that are typically analyzed in a core facility, ranging from simple protein identification to targeted analyses, and include intentional challenges to reflect realistic studies. The PRG2008 study compares different strategies for the qualitative characterization of proteins, particularly the utility of complementary methods for characterizing truncated protein forms. The use of different approaches for determining quantitative differences for several target proteins in human plasma was the focus of the PRG2009 study. The PRG2010 study explored different methods for determining specific constituents while identifying unforeseen problems that could account for unanticipated results associated with the different samples, and included (15) N-labeled proteins as an additional challenge. These studies provide a valuable educational resource to research laboratories and core facilities, as well as a mechanism for establishing good laboratory practices.

Plasma anandamide concentrations are lower in children with autism spectrum disorder.

Background: Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by restricted, stereotyped behaviors and impairments in social communication. Although the underlying biological mechanisms of ASD remain poorly understood, recent preclinical research has implicated the endogenous cannabinoid (or endocannabinoid), anandamide, as a significant neuromodulator in rodent models of ASD. Despite this promising preclinical evidence, no clinical studies to date have tested whether endocannabinoids are dysregulated in individuals with ASD. Here, we addressed this critical gap in knowledge by optimizing liquid chromatography-tandem mass spectrometry methodology to quantitatively analyze anandamide concentrations in banked blood samples collected from a cohort of children with and without ASD (N = 112). Findings: Anandamide concentrations significantly differentiated ASD cases (N = 59) from controls (N = 53), such that children with lower anandamide concentrations were more likely to have ASD (p = 0.041). In keeping with this notion, anandamide concentrations were also significantly lower in ASD compared to control children (p = 0.034). Conclusions: These findings are the first empirical human data to translate preclinical rodent findings to confirm a link between plasma anandamide concentrations in children with ASD. Although preliminary, these data suggest that impaired anandamide signaling may be involved in the pathophysiology of ASD.

Proteomics of Primary Cilia by Proximity Labeling.

While cilia are recognized as important signaling organelles, the extent of ciliary functions remains unknown because of difficulties in cataloguing proteins from mammalian primary cilia. We present a method that readily captures rapid snapshots of the ciliary proteome by selectively biotinylating ciliary proteins using a cilia-targeted proximity labeling enzyme (cilia-APEX). Besides identifying known ciliary proteins, cilia-APEX uncovered several ciliary signaling molecules. The kinases PKA, AMPK, and LKB1 were validated as bona fide ciliary proteins and PKA was found to regulate Hedgehog signaling in primary cilia. Furthermore, proteomics profiling of Ift27/Bbs19 mutant cilia correctly detected BBSome accumulation inside Ift27(-/-) cilia and revealed that ß-arrestin 2 and the viral receptor CAR are candidate cargoes of the BBSome. This work demonstrates that proximity labeling can be applied to proteomics of non-membrane-enclosed organelles and suggests that proteomics profiling of cilia will enable a rapid and powerful characterization of ciliopathies.

A double-vented tetraphasic continuous column approach to MuDPIT analysis on long capillary columns demonstrates superior proteomic coverage.

A double-vented serial tetraphasic capillary column approach is applied to proteomic MuDPIT-type analysis using extended length capillary reverse-phase columns. The heart of the tetraphasic device consists of a triphasic MuDPIT trap located upstream of a venting tee. The trap is followed by a 60 cm high-resolution capillary column. A conventional high-flow HPLC is used to develop gradients at standard flow rates and pressures. The double-vented triphasic MuDPIT trapping device relieves the capillary separation column from the salt burden during the on-line cation-exchange portion of the analysis. Two configurations are presented, a double-vented continuous column model and a discontinuous model in which the triphasic MuDPIT trap is installed on a six-port valve; both configurations were tested with 60 and 10 cm capillary columns. All four systems were challenged with a trypsin digest of undepleted human serum, and a matrix of proteomic results for the different models and column lengths are compared.