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Although peptide mass fingerprinting is currently the method of choice to identify proteins, the number of proteins available in databases is increasing constantly, and hence, the advantage of having sequence data on a selected peptide, in order to increase the effectiveness of database searching, is more crucial. Until recently, the ability to identify proteins based on the peptide sequence was essentially limited to the use of electrospray ionization tandem mass spectrometry (MS) methods. The recent development of new instruments with matrix-assisted laser desorption/ionization (MALDI) sources and true tandem mass spectrometry (MS/MS) capabilities creates the capacity to obtain high quality tandem mass spectra of peptides. In this work, using the new high resolution tandem time of flight MALDI-(TOF/TOF) mass spectrometer from Applied Biosystems, examples of successful identification and characterization of bovine heart proteins (SWISS-PROT entries: P02192, Q9XSC6, P13620) separated by two-dimensional electrophoresis and blotted onto polyvinylidene difluoride membrane are described. Tryptic protein digests were analyzed by MALDI-TOF to identify peptide masses afterward used for MS/MS. Subsequent high energy MALDI-TOF/TOF collision-induced dissociation spectra were recorded on selected ions. All data, both MS and MS/MS, were recorded on the same instrument. Tandem mass spectra were submitted to database searching using MS-Tag or were manually de novo sequenced. An interesting modification of a tryptophan residue, a "double oxidation", came to light during these analyses.

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Cell cycle transitions are generally triggered by variation in the activity of cyclin-dependent kinases (CDKs) bound to cyclins. Malaria-causing parasites have a life cycle with unique cell-division cycles, and a repertoire of divergent CDKs and cyclins of poorly understood function and interdependency. We show that Plasmodium berghei CDK-related kinase 5 (CRK5), is a critical regulator of atypical mitosis in the gametogony and is required for mosquito transmission. It phosphorylates canonical CDK motifs of components in the pre-replicative complex and is essential for DNA replication. During a replicative cycle, CRK5 stably interacts with a single Plasmodium-specific cyclin (SOC2), although we obtained no evidence of SOC2 cycling by transcription, translation or degradation. Our results provide evidence that during Plasmodium male gametogony, this divergent cyclin/CDK pair fills the functional space of other eukaryotic cell-cycle kinases controlling DNA replication.

Calcium signaling regulated by the cGMP-dependent protein kinase (PKG) controls key life cycle transitions in the malaria parasite. However, how calcium is mobilized from intracellular stores in the absence of canonical calcium channels in Plasmodium is unknown. Here, we identify a multipass membrane protein, ICM1, with homology to transporters and calcium channels that is tightly associated with PKG in both asexual blood stages and transmission stages. Phosphoproteomic analyses reveal multiple ICM1 phosphorylation events dependent on PKG activity. Stage-specific depletion of Plasmodium berghei ICM1 prevents gametogenesis due to a block in intracellular calcium mobilization, while conditional loss of Plasmodium falciparum ICM1 is detrimental for the parasite resulting in severely reduced calcium mobilization, defective egress, and lack of invasion. Our findings suggest that ICM1 is a key missing link in transducing PKG-dependent signals and provide previously unknown insights into atypical calcium homeostasis in malaria parasites essential for pathology and disease transmission.

We generated a murine monoclonal antibody (B28p) detecting an antigenic determinant shared by the immunoglobulin superfamily receptor translocationassociated 1 (IRTA1) receptor (the immunogen used to raise B28p) and an unrelated 28-kDa protein that was subsequently subjected to extensive characterization. The expression of the 28-kDa protein in normal lymphohematopoietic tissues was restricted to B cells and plasma cells and clearly differed from that expected for IRTA1 (selectively expressed by mucosa-associated lymphoid tissue [ IntroductionDifferentiation of B cells to plasma cells, in which a crucial role is played by the plasma cell transcription factors B-lymphocyteinduced maturation protein 1 (Blimp1), X-box-binding protein 1 (XBP-1), and multiple myeloma oncogene-1 protein/interferon regulatory factor 4 (MUM1/IRF4), 1,2 results in down-regulation of B-cell-associated proteins and dramatic but still poorly understood changes in the phenotypic profile. 1,2 Better understanding of these changes is relevant to the study of normal B-cell ontogenesis as well as of multiple myeloma and lymphoma subtypes characteristically associated with plasma cell differentiation. 3,4 Proteins commonly used as immunohistochemical markers of plasma cell differentiation include, in addition to intracytoplasmic immunoglobulins, CD138/syndecan-1 (a collagen-1-binding proteoglycan possibly involved in plasma cell homing properties), 5 the p63 rough endoplasmic reticulum protein (antibody VS38c), 6,7 and MUM1/IRF4 (a transcription factor expressed by a late germinal center B-cell subset and plasma cells). 8 In the course of immunizations aimed at producing a monoclonal antibody (mAb) against the intracytoplasmic portion of the human immunoglobulin superfamily receptor translocationassociated 1 (IRTA1) protein, 9 a cell surface receptor we previously showed to be selectively expressed by mucosa-associated lymphoid tissue (MALT) marginal zone B cells and monocytoid B cells, 10 we generated a murine mAb (named B28p) that detects an antigenic determinant shared by IRTA1 and an unrelated 28-kDa protein. In this paper, we describe the characteristics of this 28-kDa molecule that by 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE)/mass spectrometry was identified as the tumor protein D52 (TPD52) 11 and by immunohistochemistry showed a previously unrecognized 12,13 strong expression in all mature B cells, reaching its maximum level at the plasma cell stage. Moreover, in the Thiel myeloma cell line, TPD52 coimmunoprecipitated with annexin VI in a Ca 2ϩ -dependent manner, suggesting that these molecules may act in concert to regulate secretory processes of plasma cells, similarly to what was previously reported in pancreatic acinar cells. 14 These findings are of biologic relevance and also indicate that TPD52 can serve as a new tool for the diagnosis of B-cell malignancies. Materials and methods Generation of recombinant GST-IRTA1 proteinA cDNA fragment encoding the intracellular portion of the human IRTA1 protein was subcloned ...

Proteomic technologies based on mass spectrometry (MS) have greatly evolved in the past years, and nowadays it is possible to routinely identify thousands of peptides from complex biological samples in a single LC-MS/MS experiment. Despite the advancements in proteomic technologies, the scientific community still faces important challenges in terms of depth and reproducibility of proteomics analyses. Here, we present a multicenter study designed to evaluate long-term performance of LC-MS/MS platforms within the Spanish Proteomics Facilities Network (ProteoRed-ISCIII). The study was performed under well-established standard operating procedures, and demonstrated that it is possible to attain qualitative and quantitative reproducibility over time. Our study highlights the importance of deploying quality assessment metrics routinely in individual laboratories and in multi-laboratory studies. The mass spectrometry data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD000205.This article is part of a Special Issue entitled: HUPO 2014.

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