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Annotation and interpretation of full scan electrospray mass spectra of metabolites is complicated by the presence of a wide variety of ions. Not only protonated, deprotonated, and neutral loss ions but also sodium, potassium, and ammonium adducts as well as oligomers are frequently observed. This diversity challenges automatic annotation and is often poorly addressed by current annotation tools. In many cases, annotation is integrated in metabolomics workflows and is based on specific chromatographic peak-picking tools. We introduce mzAdan, a nonchromatography-based multipurpose standalone application that was developed for the annotation and exploration of convolved high-resolution ESI-MS spectra. The tool annotates single or multiple accurate mass spectra using a customizable adduct annotation list and outputs a list of [M+H] + candidates. MzAdan was first tested with a collection of 408 analytes acquired with flow injection analysis. This resulted in 402 correct [M+H] + identifications and, with combinations of sodium, ammonium, and potassium adducts and water and ammonia losses within a tolerance of 10 mmu, explained close to 50% of the total ion current. False positives were monitored with mass accuracy and bias as well as chromatographic behavior which led to the identification of adducts with calcium instead of the expected potassium. MzAdan was then integrated in a workflow with XCMS for the untargeted LC-MS data analysis of a 52 metabolite standard mix and a human urine sample. The results were benchmarked against three other annotation tools, CAMERA, findMAIN, and CliqueMS: findMAIN and mzAdan consistently produced higher numbers of [M+H] + candidates compared with CliqueMS and CAMERA, especially with co-eluting metabolites. Detection of low-intensity ions and correct grouping were found to be essential for annotation performance. Graphical abstract.

Data-independent acquisition (DIA) offers several advantages over data-dependent acquisition (DDA) schemes for characterizing complex protein digests analyzed by LC-MS/MS. In contrast to the sequential detection, selection, and analysis of individual ions during DDA, DIA systematically parallelizes the fragmentation of all detectable ions within a wide m/z range regardless of intensity, thereby providing broader dynamic range of detected signals, improved reproducibility for identification, better sensitivity, and accuracy for quantification, and, potentially, enhanced proteome coverage. To fully exploit these advantages, composite or multiplexed fragment ion spectra generated by DIA require more elaborate processing algorithms compared to DDA. This review examines different DIA schemes and, in particular, discusses the concepts applied to and related to data processing. Available software implementations for identification and quantification are presented as comprehensively as possible and examples of software usage are cited. Processing workflows, including complete proprietary frameworks or combinations of modules from different open source data processing packages are described and compared in terms of software availability and usability, programming language, operating system support, input/output data formats, as well as the main principles employed in the algorithms used for identification and quantification. This comparative study concludes with further discussion of current limitations and expectable improvements in the short- and midterm future., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In this chapter, we describe the use of SuperHirn and MSight, two complementary tools developed to the processing of label-free LC/MS data in view of the quantitation of proteomics samples. While MSight is mainly dedicated to the visualisation and navigation into LC/MS data, SuperHirn is specialised in peak detection, normalisation and alignment of LC/MS runs. These two tools can be used in a complementary way and one of the possible usages is described here.

The cellular abundance of transcription factors (TFs) is an important determinant of their regulatory activities. Deriving TF copy numbers is therefore crucial to understanding how these proteins control gene expression. We describe a sensitive selected reaction monitoring-based mass spectrometry assay that allowed us to determine the copy numbers of up to ten proteins simultaneously. We applied this approach to profile the absolute levels of key TFs, including PPARγ and RXRα, during terminal differentiation of mouse 3T3-L1 pre-adipocytes. Our analyses revealed that individual TF abundance differs dramatically (from ∼250 to >300,000 copies per nucleus) and that their dynamic range during differentiation can vary up to fivefold. We also formulated a DNA binding model for PPARγ based on TF copy number, binding energetics and local chromatin state. This model explains the increase in PPARγ binding sites during the final differentiation stage that occurs despite a concurrent saturation in PPARγ copy number. [ABSTRACT FROM AUTHOR]

A Correction to this paper has been published: https://doi.org/10.1007/s00216-020-03148-9 [ABSTRACT FROM AUTHOR]

Dendritic cells (DCs) are specialized leukocytes that orchestrate the adaptive immune response. Mass spectrometry (MS)-based proteomic study of these cells presents technical challenges, especially when the DCs are human in origin due to the paucity of available biological material. Here, to maximize MS coverage of the global human DC proteome, different cell disruption methods, lysis conditions, protein precipitation, and protein pellet solubilization and denaturation methods were compared. Mechanical disruption of DC cell pellets under cryogenic conditions, coupled with the use of RIPA (radioimmunoprecipitation assay) buffer, was shown to be the method of choice based on total protein extraction and on the solubilization and identification of nuclear proteins. Precipitation by acetone was found to be more efficient than that by 10% trichloroacetic acid (TCA)/acetone, allowing in excess of 28% more protein identifications. Although being an effective strategy to eliminate the detergent residue, the acetone wash step caused a loss of protein identifications. However, this potential drawback was overcome by adding 1% sodium deoxycholate into the dissolution buffer, which enhanced both solubility of the precipitated proteins and digestion efficiency. This in turn resulted in 6 to 11% more distinct peptides and 14 to 19% more total proteins identified than using 0.5 M triethylammonium bicarbonate alone, with the greatest increase (34%) for hydrophobic proteins. [ABSTRACT FROM AUTHOR]

Abstract: Leishmaniasis is a vector-borne disease caused by the protozoa Leishmania. We have analyzed and compared the sequences of three experimental exoproteomes of Leishmania promastigotes from different species to determine their specific features and to identify new candidate proteins involved in interactions of Leishmania with the host. The exoproteomes differ from the proteomes by a decrease in the average molecular weight per protein, in disordered amino acid residues and in basic proteins. The exoproteome of the visceral species is significantly enriched in sites predicted to be phosphorylated as well as in features frequently associated with molecular interactions (intrinsic disorder, number of disordered binding regions per protein, interaction and/or trafficking motifs) compared to the other species. The visceral species might thus have a larger interaction repertoire with the host than the other species. Less than 10% of the exoproteomes contain heparin-binding and RGD sequences, and ~30% the host targeting signal RXLXE/D/Q. These latter proteins might thus be exported inside the host cell during the intracellular stage of the infection. Furthermore we have identified nine protein families conserved in the three exoproteomes with specific combinations of Pfam domains and selected eleven proteins containing at least three interaction and/or trafficking motifs including two splicing factors, phosphomannomutase, 2,3-bisphosphoglycerate-independent phosphoglycerate mutase, the paraflagellar rod protein-1D and a putative helicase. Their role in host–Leishmania interactions warrants further investigation but the putative ATP-dependent DEAD/H RNA helicase, which contains numerous interaction motifs, a host targeting signal and two disordered regions, is a very promising candidate. [Copyright &y& Elsevier]