Do we want our data raw? Including binary mass spectrometry data in public proteomics data repositories

With the human Plasma Proteome Project (PPP) pilot phase completed, the largest and most ambitious proteomics experiment to date has reached its first milestone. The correspondingly impressive amount of data that came from this pilot project emphasized the need for a centralized dissemination mechanism and led to the development of a detailed, PPP specific data gathering infrastructure at the University of Michigan, Ann Arbor as well as the protein identifications database project at the European Bioinformatics Institute as a general proteomics data repository. One issue that crept up while discussing which data to store for the PPP concerns whether the raw, binary data coming from the mass spectrometers should be stored, or rather the more compact and already significantly processed peak lists. As this debate is not restricted to the PPP but relates to the proteomics community in general, we will attempt to detail the relative merits and caveats associated with centralized storage and dissemination of raw data and/or peak lists, building on the extensive experience gained during the PPP pilot phase. Finally, some suggestions are made for both immediate and future storage of MS data in public repositories.     

QuantiSpec — Quantitative mass spectrometry data analysis of 15N-metabolically labeled proteins

For relative protein quantitation by mass spectrometry we metabolically labeled E. coli bacteria with ¹⁵N-enriched diets. Proteins extracted from ¹⁵N-labeled and unlabeled E. coli bacteria were mixed, separated by two-dimensional gel electrophoresis and enzymatically digested. The resulting tryptic peptides were analyzed by MALDI mass spectrometry. For the relative protein quantitation we developed fully automated software, QuantiSpec (Quantitative Mass Spectrometry Analysis Software), which uses data from MALDI TOF mass spectrometry and the Mascot database search engine. QuantiSpec detects natural as well as partially or fully labeled peptide isotope distributions. For each identified peptide the ¹⁵N incorporation rate is determined by comparing the experimental to a set of theoretical isotope patterns based on the peptide sequence. Relative quantitation is accomplished by calculating the signal intensity ratios for each ¹⁴N/¹⁵N peptide pair.     

A targeted metabolomic procedure for amino acid analysis in different biological specimens by ultra-high-performance liquid chromatography–tandem mass spectrometry

Introduction

Amino acid analysis in biological fluids is essential for the study of inborn errors of metabolism (IEM) and other diseases.

Objectives

Our aim was to develop a UPLC-MS/MS procedure for the analysis of 25 amino acids and identification of 17 related compounds.

Methods

Sample treatment conditions were optimized for plasma, urine, cerebrospinal fluid (CSF) and dried blood spots. Amino acids and related compounds were analyzed on a Waters ACQUITY UPLC H-class instrument with a reversed-phase C-18 column using water and acetonitrile with 0.1% formic acid as the mobile phases (run time?=?9 min). The detection was performed with a Waters Xevo TQD triple-quadrupole mass spectrometer using positive electrospray ionization in the multiple reaction monitoring mode.

Results

The method linearity, intra-assay and inter-assay precision, detection limit, quantification limit and trueness analysis displayed adequate results in both physiological and pathological conditions. Method comparison was performed between UPLC-MS/MS and ion exchange chromatography (IEC) with ninhydrin derivatization, and the methods showed good agreement, except for 4-hydroxyproline, aspartate and citrulline. Paediatrics age-related reference values in plasma, urine and CSF were established and patients with different IEM were easily identified.

Conclusion

We report a modified UPLC-MS/MS procedure for the analysis of 42 amino acids and related compounds in different specimens. The method is fast, sensitive and robust, and it has been validated to be an alternative to the traditional IEC procedure as the routine method used in metabolic laboratories. The method greatly decreases the run time of the analysis while displaying good metrological results.

     

What place for polyacrylamide in proteomics?

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) continues to deliver high quality protein resolution and dynamic range for the proteomics researcher. To remain as the preferred method for protein separation and characterization, several key steps need to be implemented to ensure quality sample preparation and speed of analysis. Here, we describe the progress made towards establishing 2D-PAGE as the optimal separation tool for proteomics research.     

What is the best age to circumcise? A medical and ethical analysis

Circumcision is often claimed to be simpler, safer and more cost-effective when performed in the neonatal period as opposed to later in life, with a greater benefit-to-risk ratio. In the first part of this paper, we critically examine the evidence base for these claims, and find that it is not as robust as is commonly assumed. In the second part, we demonstrate that, even if one simply grants these claims for the sake of argument, it still does not follow that neonatal circumcision is ethically permissible absent urgent medical necessity. Based on a careful consideration of the relevant evidence, arguments and counterarguments, we conclude that medically unnecessary penile circumcision—like other medically unnecessary genital procedures, such as ‘cosmetic’ labiaplasty—should not be performed on individuals who are too young (or otherwise unable) to provide meaningful consent to the procedure.     

Quantitative analysis of lignocaine and metabolites in equine urine and plasma by liquid chromatography–tandem mass spectrometry

In this paper, a method for the sensitive and reproducible analysis of lignocaine and its four principal metabolites, monoethylxylidide (MEGX), glycylxylidide (GX), 3-hydroxylignocaine (3-HO-LIG), 4-hydroxylignocaine (4-HO-LIG) in equine urine and plasma samples is presented. The method uses liquid chromatography coupled to tandem mass spectrometry operating in electrospray ionisation positive ion mode (+ESI) via multiple reaction monitoring (MRM). Sample preparation involved solid-phase extraction using a mixed-mode phase. The internal standard adopted was lignocaine-d₁₀. Lignocaine and its metabolites were successfully resolved using an octadecylsilica reversed-phase column using a gradient mobile phase of acetonitrile and 0.1% (v/v) aqueous formic acid at a flow rate of 300 μL/min. Target analytes and the internal standard were determined by using the following transitions; lignocaine, 235.2 > 86.1; 3-HO-LIG and 4-HO-LIG, 251.2 > 86.1; MEGX, 207.1 > 58.1; GX, 179.1 > 122.1; and lignocaine-d₁₀, 245.2 > 96.1. Calibration curves were generated over the range 1–100 ng/mL for plasma samples and 1–1000 ng/mL for urine samples. The method was validated for instrument linearity, repeatability and detection limit (IDL), method linearity, repeatability, detection limit (MDL), quantitation limit (LOQ) and recovery. The method was successfully used to analyse both plasma and urine samples following a subcutaneous administration of lignocaine to a thoroughbred horse.     

Gas chromatography–mass spectrometry analysis of fatty acid profiles of Antarctic and non-Antarctic yeasts

The fatty acid profiles of Antarctic (n = 7) and non-Antarctic yeasts (n = 7) grown at different temperatures were analysed by gas chromatography–mass spectrometry. The Antarctic yeasts were enriched in oleic 18:1 (20–60 %), linoleic 18:2 (20–50 %) and linolenic 18:3 (5–40 %) acids with lesser amounts of palmitic 16:0 (<15 %) and palmitoleic 16:1 (<10 %) acids. The non-Antarctic yeasts (n = 4) were enriched in 18:1 (20–55 %, with R. mucilaginosa at 75–80 %) and 18:2 (10–40 %) with lesser amounts of 16:0 (<20 %), 16:1 (<20 %) and stearic 18:0 (<10 %) acids. By contrast, Saccharomyces cerevisiae strains (n = 3) were enriched in 16:1 (30–50 %) and 18:1 (20–40 %) with lesser amounts of 16:0 (10–25 %) and 18:0 (5–10 %) acids. Principal component analysis grouped the yeasts into three clusters, one belonging to the S. cerevisiae strains (enriched in 16:0, 16:1 and 18:1), one to the other non-Antarctic yeasts (enriched in 18:1 and 18:2) and the third to the Antarctic yeasts (enriched in 18:2 and 18:3).     

Is mass spectrometry ready for proteome-wide protein expression analysis?

Recent advances in mass spectrometry will soon allow routine analysis of protein expression levels. How close are we to true quantitative proteomics?     

What predicts religiosity? A multinational analysis of reproductive and cooperative morals

Theories of the sources of contemporary individual differences in religiosity have been proposed involving religiosity’s role both in (1) enhancing within-group cooperation and (2) supporting high-commitment reproductive strategies. The present study used data from 296,959 individuals in around 90 countries from the World Values Survey/European Values Study to test the relative strength of individual differences in cooperative morals and reproductive morals in predicting individual differences in religiosity. Cooperative morals tended not to predict religiosity either substantially or in a consistent direction across world regions when entered simultaneously with reproductive morals. In contrast, more-restrictive reproductive morals were significant predictors of increased religiosity in every region, with the size of the relationship being small in poorer regions and large in wealthier regions. These findings run counter to the view that religiosity has a fundamental connection with cooperative morals; instead, particularly in developed countries, individuals’ relationships with religious groups are more closely aligned with reproductive strategies.     

Accurate analysis of urea in milk and milk powder by isotope dilution gas chromatography–mass spectrometry

A high order method for measuring urea concentrations in milk and milk powder was developed. The method can be applied to certify the concentration of urea in some new milk and milk powder CRMs. This high accurate method for analysis of milk is valuable given the inherent challenges associated with the complexity of the sample matrix. A measurement procedure based on gas chromatography/isotope dilution mass spectrometry (GC/IDMS) was developed. Samples were pre-treated with acetonitrile to remove proteins and the method was applied to determine urea concentrations in milk and milk powder. Excellent precision was obtained, with within- and between-set coefficients of variation of 0.15–0.46 and 0.18–0.65%, respectively. The measurement uncertainty is evaluated. The method can trace to mass.     

Clinical applications of mass spectrometry-based proteomics in cancer: Where are we?

Tumor tissue processing methodologies in combination with data-independent acquisition mass spectrometry (DIA-MS) have emerged that can comprehensively analyze the proteome of multiple tumor samples accurately and reproducibly. Increasing recognition and adoption of these technologies has resulted in a tranche of studies providing novel insights into cancer classification systems, functional tumor biology, cancer biomarkers, treatment response and drug targets. Despite this, with some limited exceptions, MS-based proteomics has not yet been implemented in routine cancer clinical practice. Here, we summarize the use of DIA-MS in studies that may pave the way for future clinical cancer applications, and highlight the role of alternative MS technologies and multi-omic strategies. We discuss limitations and challenges of studies in this field to date and propose steps for integrating proteomic data into the cancer clinic.     

Probing the stoichiometry of β2-adrenergic receptor phosphorylation by targeted mass spectrometry

Background

Protein phosphorylation of G-protein-coupled receptors (GPCR) is central to the myriad of functions that these ubiquitous receptors perform in biology. Although readily addressable with the use of phospho-specific antibodies, analysis phosphorylation at the level of stoichiometry requires receptor isolation and advanced proteomics. We chose two key sites of potential phosphorylation of human beta2-adrenergic receptor (β2AR residues S355 and S356) to ascertain the feasibility of applying targeted mass spectrometry to establishing the stoichiometry of the phosphorylation.

Method

We stimulated HEK293 cells stably expressing Flag-tagged β2AR-eGFP with 10 μM beta-adrenergic agonist (isoproterenol) and made use of proteomics and targeted mass spectrometry (MS) to quantify the molar ration of phosphorylation on S355 and S356 versus non-phosphorylated receptor in agonist-treated cells.

Results

Phosphorylation of either S355 or S356 residue occurred only for agonist-occupied β2AR. The results demonstrated that pS356 is the dominant site of protein phosphorylation. The abundance of the p356 was 8.6-fold more than that of pS355. Calculation of the molar ratio of phosphorylated (pS355 plus pS356) versus non-phosphorylated receptor reveals that at high occupancy of the receptor only 12.4% of the β2AR is phosphorylated at these sites.

Conclusions

Application of advanced proteomics and use of the most sensitive targeted MS strategy makes possible the detection and quantification of phosphorylation of very low abundance peptide digests of β2AR. Establishing the stoichiometry of two key sites of agonist-stimulated phosphorylation with β2AR is an essential first-step to global analysis of the stoichiometry of GPCR phosphorylation.     

What is Causal Specificity About,and What is it Good for in Philosophy of Biology?

Acta Biotheoretica - The concept of causal specificity is drawing considerable attention from philosophers of biology. It became the rationale for rejecting (and occasionally, accepting) a thesis...