Nucleotide-Induced Membrane-Proximal Proteolysis Controls the Substrate Specificity of T Cell Ecto-ADP-Ribosyltransferase ARTC2.2.

ARTC2.2 is a toxin-related, GPI-anchored ADP-ribosyltransferase expressed by murine T cells. In response to NAD(+) released from damaged cells during inflammation, ARTC2.2 ADP-ribosylates and thereby gates the P2X7 ion channel. This induces ectodomain shedding of metalloprotease-sensitive cell surface proteins. In this study, we show that ARTC2.2 itself is a target for P2X7-triggered ectodomain shedding. We identify the metalloprotease cleavage site 3 aa upstream of the predicted GPI anchor attachment site of ARTC2.2. Intravenous injection of NAD(+) increased the level of enzymatically active ARTC2.2 in serum, indicating that this mechanism is operative also under inflammatory conditions in vivo. Radio-ADP-ribosylation assays reveal that shedding refocuses the target specificity of ARTC2.2 from membrane proteins to secretory proteins. Our results uncover nucleotide-induced membrane-proximal proteolysis as a regulatory mechanism to control the substrate specificity of ARTC2.2.

Monitoring of protease catalyzed reactions by quantitative MALDI MS using metal labeling.

Quantitative mass spectrometry is a powerful tool for the determination of enzyme activities as it does not require labeled substrates and simultaneously allows for the identification of reaction products. However, major restrictions are the limited number of samples which can be measured in parallel due to the need for isotope labeled internal standards. Here we describe the use of metal labeling of peptides for the setup of multiplexed enzyme activity assays. After proteolytic reaction, using the protease trypsin, remaining substrates and peptide products formed in the reaction were labeled with metal chelators complexing rare earth metal ions. Labeled peptides were quantified with high accuracy and over a wide dynamic range (at least 2 orders of magnitude) using MALDI MS in case of simple peptide mixtures or by LC-MALDI MS for complex substrate mixtures and used for the monitoring of time-dependent product formation and substrate consumption. Due to multiplexing capabilities and accuracy, the presented approach will be useful for the determination of enzyme activities with a wide range of biochemical and biotechnological applications.

Metal labeling for accurate multiplexed peptide quantification via matrix-assisted laser desorption/ionization mass spectrometry.

Two peptide quantification strategies, the isobaric tags for relative or absolute quantitation (iTRAQ) labeling methodology and a metal-chelate labeling approach, were compared using matrix-assisted laser desorption/ionization-TOF/TOF MS and MS/MS analysis. Amino- and cysteine-directed labeling using the rare earth metal chelator 1,4,7,10-tetraazacyclododecane-N,N',N″,N″'-tetraacetic acid (DOTA) were applied for relative quantification of single peptides and a six-protein mixture. For analyte ratios close to one, iTRAQ and amino-directed DOTA labeling delivered overall comparable results regarding accuracy and reproducibility. In contrast, the MS-based quantification via amino-directed lanthanide-DOTA tags was more accurate for analyte ratios ≥5 and offered an extended dynamic range of three orders of magnitude. Our results show that the amino-directed DOTA labeling is an alternative relative quantification tool offering advantages like flexible multiplexing possibilities and, in particular, large dynamic ranges, which should be useful in sophisticated, targeted issues, where the accurate determination of extremely different protein or peptide concentration becomes relevant.

Improved reporter ion assignment of raw isobaric stable isotope labeled liquid chromatography/matrix-assisted laser desorption/ionization tandem time-of-flight mass spectral data for quantitative proteomics.

RATIONALE: Isobaric labeling strategies (e.g. iTRAQ or TMT) are commonly applied in tandem mass spectrometric (MS/MS) level quantitative proteomics. However, we frequently observed missing isotope reporter ion signals in a large-scale liquid chromatography/matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometric (LC/MALDI-TOF/TOF) quantitative proteomics experiment. To understand this issue, we systematically investigated the processing of MS/MS spectra into peak lists prior to peptide identification and quantification. METHODS: A 15-protein standard, with six proteins in different concentrations, was labeled with iTRAQ 4-plex, iTRAQ 8-plex or TMT 6-plex, tryptic digested and measured using LC/MALDI-TOF/TOF. Three commercially and open-source available peak list generation software tools were compared based on missing reporter ions, peptide identification and quantification. RESULTS: We found that each tool discarded lower-intensity reporter ions, when they followed a higher intensity reporter ion, due to the implemented de-isotoping algorithms. By using the non-de-isotoping setting within TS2Mascot, we found that all reporter ions are exported, yet less peptides were identified with Mascot. Therefore, we developed a strategy merging the de-isotoped and non-de-isotoped outputs from TS2Mascot using the Perl script RICmerge.pl. CONCLUSIONS: With this approach, we correctly quantified all labeled peptides that were identified within the 15-protein standard. This strategy allows improved annotation of isobaric tag labeled peptide MS/MS spectra and improves downstream peptide and protein quantification in proteomics studies.

Isotope pattern deconvolution for peptide mass spectrometry by non-negative least squares/least absolute deviation template matching.

BACKGROUND: The robust identification of isotope patterns originating from peptides being analyzed through mass spectrometry (MS) is often significantly hampered by noise artifacts and the interference of overlapping patterns arising e.g. from post-translational modifications. As the classification of the recorded data points into either 'noise' or 'signal' lies at the very root of essentially every proteomic application, the quality of the automated processing of mass spectra can significantly influence the way the data might be interpreted within a given biological context. RESULTS: We propose non-negative least squares/non-negative least absolute deviation regression to fit a raw spectrum by templates imitating isotope patterns. In a carefully designed validation scheme, we show that the method exhibits excellent performance in pattern picking. It is demonstrated that the method is able to disentangle complicated overlaps of patterns. CONCLUSIONS: We find that regularization is not necessary to prevent overfitting and that thresholding is an effective and user-friendly way to perform feature selection. The proposed method avoids problems inherent in regularization-based approaches, comes with a set of well-interpretable parameters whose default configuration is shown to generalize well without the need for fine-tuning, and is applicable to spectra of different platforms. The R package IPPD implements the method and is available from the Bioconductor platform (http://bioconductor.fhcrc.org/help/bioc-views/devel/bioc/html/IPPD.html).

Cationic detergents enable the separation of membrane proteins of Plasmodium falciparum-infected erythrocytes by 2D gel electrophoresis.

The intraerythrocytic stage of Plasmodium falciparum alters the characteristics of its host cell by exporting selected plasmodial proteins. Although it is clear that the physicochemical and immunobiological properties of the host cell are modulated during parasite development, the involved plasmodial proteins and their mode of action are not completely known. Using cetyltrimethylammonium bromide (CTAB) or benzyldimethyl-n-hexadecylammonium chloride (16-BAC) for the first dimension and SDS for the second dimension, we separated proteins from membranes of human erythrocytes and of erythrocytes infected with the malaria parasite P. falciparum. Protein spots were analyzed by MALDI-TOF/TOF MS and annotated in respective 2D master gels. By using the alternative 2D approach, characteristic host cell membrane proteins and, more importantly, membrane-associated and exported plasmodial proteins were identified that might play a role in parasite-induced host cell modulation.

Quantitative protease cleavage site profiling using tandem-mass-tag labeling and LC-MALDI-TOF/TOF MS/MS analysis.

Knowledge of cleavage site specificity and activity are major prerequisites for understanding protease function. On the basis of a recently presented approach for proteomic identification of cleavage sites (PICS) in proteome-derived peptide libraries, we developed an isobaric labeling quantitative LC-MALDI-TOF/TOF MS/MS approach (Q-PICS) for simultaneous determination of cleavage site specificity and robust relative quantification of proteolytic events. For GluC-protease, 737 cleavage sites were identified in a yeast proteome-derived peptide library; 94.0% showed the typical GluC specificity for peptide bonds at glutamyl and aspartyl residues. The six-plex tandem mass tagging strategy allowed for three simultaneous replicates in a single run, guaranteeing high confidence and robust statistics for quantitative measurements. Using the quantitative capacity of Q-PICS, we performed a comparison of cleavage site specificity of GluC in two different buffer systems. The results support earlier findings describing that apparent difference between the buffer systems are probably caused by the inhibitory effect of bicarbonate on the overall GluC activity and that the preference for Glu-X bonds compared to Asp-X bonds is independent of the buffer system used.

Quantification of the Fabry marker lysoGb3 in human plasma by tandem mass spectrometry.

Morbus Fabry is a hereditary metabolic disorder with low prevalence and late clinical manifestation. A defect in the α-galactosidase gene leads to lysosomal accumulation of the glycolipid globotriaosylceramide (Gb3). Gb3 may be used for monitoring of enzyme replacement therapy (ERT), but diagnostic sensitivity is limited. Recently, globotriaosylsphingosine (lysoGb3) was introduced as a promising new marker with significantly better sensitivity. For Fabry diagnosis, clinical studies and possible therapy monitoring, we established a fast and reliable LC-MS/MS assay for quantification of lysoGb3 in human plasma. Protein precipitation and glycolipid extraction from EDTA plasma was performed using acetone/methanol. Samples were analyzed by UPLC-MS/MS in MRM mode. In contrast to HPLC with fluorescence detection, the LC-MS/MS method requires no derivatization, less sample preparation and less instrument analysis time (<3 min). As internal standard (ISTD), a glycine derivative of lysoGb3 was synthesized, and the product was purified by HPLC. ISTD properties such as polarity (affecting extraction and elution), ionization and fragmentation pathway were almost identical compared to the analyte. The new LC-MS/MS assay for the Fabry marker lysoGb3 shows good performance and allowed for better discrimination between Fabry patients and controls than Gb3.

Rapid selection of peptide containing fractions in off-line 2-D HPLC in shotgun proteome analysis by screening with MALDI TOF MS.

A simple and fast screening method for the selection of fractions of first dimension separation to be analyzed in second dimension-MS/MS experiments in offline multidimensional liquid chromatographic separation schemes for shotgun proteome analysis was developed. The method is based on the measurement of total peptide content of the first dimension fractions by MALDI MS and was established using a tryptic digest of a bacterial proteome. The results of the screening process were in good agreement with those obtained in a detailed proteome analysis performed by RPxion-pair RP-MALDI TOF/TOF MS analysis. The method supports a straightforward planning of experiments, also enabling a reduction of overall measurement time in shotgun proteome analysis.