Despite major advantages in the field of proteomics, the analysis of PTMs still poses a major challenge; thus far, preventing insights into the role and regulation of protein networks. Additionally, ...top-down sequencing of proteins is another powerful approach to reveal comprehensive information for biological function. A commonly used fragmentation technique in MS-based peptide sequencing is CID. As CID often fails in PTM-analysis and performs best on doubly-charged, short and middle-sized peptides, confident peptide identification may be hampered. A newly developed fragmentation technique, namely electron transfer dissociation (ETD), supports both, PTM- and top-down analysis, and generally results in more confident identification of long, highly charged or modified peptides. The following review presents the theoretical background of ETD and its technical implementation in mass analyzers. Furthermore, current improvements of ETD and approaches for the PTM-analysis and top-down sequencing are introduced. Alternating both fragmentation techniques, ETD and CID, increases the amount of information derived from peptide fragmentation, thereby enhancing both, peptide sequence coverage and the confidence of peptide and protein identification.
Background:
C-type natriuretic peptide (CNP)/natriuretic peptide receptor 2 (NPR2) signaling is essential for long bone growth. Enhanced CNP production caused by chromosomal translocations results in ...tall stature, a Marfanoid phenotype, and skeletal abnormalities. A similar phenotype was described in a family with an activating NPR2 mutation within the guanylyl cyclase domain.
Case:
Here we describe an extremely tall male without skeletal deformities, with a novel NPR2 mutation (p.Arg655Cys) located in the kinase homology domain.
Objectives:
The objective of the study was to investigate the functional and structural effects of the NPR2 mutation.
Methods:
Guanylyl cyclase activities of wild-type vs mutant NPR2 were analyzed in transfected human embryonic kidney 293 cells and in skin fibroblasts. The former were also used to study possible interactions between both isoforms. Homology modeling was performed to understand the molecular impact of the mutation.
Results:
CNP-stimulated cGMP production by the mutant NPR2 was markedly increased in patient skin fibroblasts and transfected human embryonic kidney 293 cells. The stimulatory effects of ATP on CNP-dependent guanylyl cyclase activity were augmented, suggesting that this novel mutation enhances both the responsiveness of NPR2 to CNP and its allosteric modulation/stabilization by ATP. Coimmunoprecipitation showed that wild-type and mutant NPR2 can form stable heterodimers, suggesting a dominant-positive effect. In accordance with augmented endogenous receptor activity, plasma N-terminal pro-CNP (a marker of CNP production in tissues) was reduced in the proband.
Conclusions:
We report the first activating mutation within the kinase homology domain of NPR2, resulting in extremely tall stature. Our observations emphasize the important role of this domain in the regulation of guanylyl cyclase activity and bone growth in response to CNP.
In analytical sciences, there is a general need for quality control to assess whether a product or a process meets defined requirements. Especially in proteomics, which implies analysis of ten ...thousands of analytes within a complex mixture, quality control to validate LC-MS performance and method setup is inevitable to achieve day-to-day-, inter-system-, as well as inter-user reproducibility. Thus, results deriving from LC-MS analyses can be benchmarked and the need for system maintenance can be revealed. In particular with the advent of label-free quantification of peptides and proteins, which above all depends on highly stable and reproducible LC separations, HPLC performance has to be appropriately monitored throughout the entire analytical procedure to assure quality and validity of the obtained data. Oftentimes, proteolytic digests of standard proteins are used in this context; however, this approach implies some limitations, such as inadequate batch-to-batch reproducibility, limited (if any) dynamic range and compositional inflexibility. Here, we present an alternative strategy of nano-LC-MS/MS quality control based on a mixture of synthetic peptides covering the entire LC-gradient as well as a dynamic range of more than two orders of magnitude. Thus, (i) reproducibility of LC separation, (ii) MS performance (including limit of detection, identification and quantification), as well as (iii) overall nano-LC-MS system performance and reproducibility can be routinely monitored even in highly complex samples.
Store-operated Ca2+ entry (SOCE) is the major route of Ca2+ influx in platelets. The Ca2+ sensor stromal interaction molecule 1 (STIM1) triggers SOCE by forming punctate structures with the Ca2+ ...channel Orai1 and the inositol trisphosphate receptor (IP3R), thereby linking the endo-/sarcoplasmic reticulum to the plasma membrane. Here, we identified the BAR domain superfamily member bridging integrator 2 (BIN2) as an interaction partner of STIM1 and IP3R in platelets. Deletion of platelet BIN2 (Bin2fl/fl,Pf4-Cre mice) resulted in reduced Ca2+ store release and Ca2+ influx in response to all tested platelet agonists. These defects were a consequence of impaired IP3R function in combination with defective STIM1-mediated SOC channel activation, while Ca2+ store content and agonist-induced IP3 production were unaltered. This severely defective Ca2+ signaling translated into impaired thrombus formation under flow and a protection of Bin2fl/fl,Pf4-Cre mice in models of arterial thrombosis and stroke. Our results establish BIN2 as a central regulator of platelet activation in thrombosis and thrombo-inflammatory disease settings.
Yeast proteome research comprises two different aspects: with respect to systemic fungal infections (fungemias), invasive candidiasis, for instance by Candida albicans, is among the most common ...causes of morbidity and mortality particularly in the expanding population of immunocompromised patients, which rises a high medical and pharmaceutical interest in this facultative pathogenic organism. Apart from its clinical relevance, yeast research moreover provides an indispensable source of knowledge regarding fundamental biochemical processes of eukaryotic cells. In this context, the budding yeast Saccharomyces cerevisiae is, in addition to its multiple industrial applications, one of the most extensively used microorganisms and serves as the best understood eukaryotic model system so far. Consequently, numerous studies have focused on gaining insight into the yeast proteome, with protein MS providing a very efficient technology to cope with this task since it enables both protein identification and differential quantification of cellular material. In this review we present an overview of recent advances in yeast organelle and membrane proteomics focusing on the cell wall, plasma membrane, mitochondria and vacuole.
Blood platelets are key players standing at the crossroads between physiologically occurring hemostasis and pathologic thrombus formation. As these cellular particles lack a nucleus, intra- and ...intercellular processes involved in platelet activity and function are almost exclusively regulated on the protein level. In particular, posttranslational protein modification by phosphorylation, which allows for a quick and highly dynamic transduction of cellular signals, is discussed in this context. In addition, since platelet activation and aggregation usually require surface contact with the surrounding tissue, special interest focuses on this contacting region, and hence on the subproteome of the platelet plasma membrane. In this chapter, we present a mass spectrometry-driven approach capable of dealing with the task of platelet plasma membrane proteomics and phosphoproteomics. The outlined protocols include strategies for the isolation and purification of plasma membrane proteins by aqueous two-phase partitioning and subsequent enrichment of phosphopeptides via titanium dioxide chromatography.
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