Mitochondrial CLPP2 Assists Coordination and Homeostasis of Respiratory Complexes.

Protein homeostasis in eukaryotic organelles and their progenitor prokaryotes is regulated by a series of proteases including the caseinolytic protease (CLPP). CLPP has essential roles in chloroplast biogenesis and maintenance, but the significance of the plant mitochondrial CLPP remains unknown and factors that aid coordination of nuclear- and mitochondrial-encoded subunits for complex assembly in mitochondria await discovery. We generated knockout lines of the single gene for the mitochondrial CLP protease subunit, CLPP2, in Arabidopsis (Arabidopsis thaliana). Mutants showed a higher abundance of transcripts from mitochondrial genes encoding oxidative phosphorylation protein complexes, whereas nuclear genes encoding other subunits of the same complexes showed no change in transcript abundance. By contrast, the protein abundance of specific nuclear-encoded subunits in oxidative phosphorylation complexes I and V increased in CLPP2 knockouts, without accumulation of mitochondrial-encoded counterparts in the same complex. Complexes with subunits mainly or entirely encoded in the nucleus were unaffected. Analysis of protein import and function of complex I revealed that while function was retained, protein homeostasis was disrupted, leading to accumulation of soluble subcomplexes of nuclear-encoded subunits. Therefore, CLPP2 contributes to the mitochondrial protein degradation network through supporting coordination and homeostasis of protein complexes encoded across mitochondrial and nuclear genomes.

Systematic and quantitative comparison of digest efficiency and specificity reveals the impact of trypsin quality on MS-based proteomics.

Trypsin is the most frequently used proteolytic enzyme in mass spectrometry-based proteomics. Beside its good availability, it also offers some major advantages such as an optimal average peptide length of ~14 amino acids, and typically the presence of at least two defined positive charges at the N-terminus as well as the C-terminal Arg/Lys, rendering tryptic peptides well suited for CID-based LC-MS/MS. Here, we conducted a systematic study of different types of commercially available trypsin in order to qualitatively and quantitatively compare cleavage specificity, efficiency as well as reproducibility and the potential impact on quantitation and proteome coverage. We present a straightforward strategy applied to complex digests of human platelets, comprising (1) digest controls using a monolithic column HPLC-setup, (2) SCX enrichment of semitryptic/nonspecific peptides, (3) targeted MRM analysis of corresponding full cleavage/missed cleavage peptide pairs as well as (4) LC-MS analyses of complete digests with a three-step data interpretation. Thus, differences in digest performance can be readily assessed, rendering these procedures extremely beneficial to quality control not only the trypsin of choice, but also to effectively compare as well as optimize different digestion conditions and to evaluate the reproducibility of a dedicated digest protocol for all kinds of quantitative proteome studies.

Global analysis of the mitochondrial N-proteome identifies a processing peptidase critical for protein stability.

Many mitochondrial proteins are synthesized with N-terminal presequences that are removed by specific peptidases. The N-termini of the mature proteins and thus peptidase cleavage sites have only been determined for a small fraction of mitochondrial proteins and yielded a controversial situation for the cleavage site specificity of the major mitochondrial processing peptidase (MPP). We report a global analysis of the N-proteome of yeast mitochondria, revealing the N-termini of 615 different proteins. Significantly more proteins than predicted contained cleavable presequences. We identified the intermediate cleaving peptidase Icp55, which removes an amino acid from a characteristic set of MPP-generated N-termini, solving the controversial situation of MPP specificity and suggesting that Icp55 converts instable intermediates into stable proteins. Our results suggest that Icp55 is critical for stabilization of the mitochondrial proteome and illustrate how the N-proteome can serve as rich source for a systematic analysis of mitochondrial protein targeting, cleavage and turnover.

Analysis of the membrane proteome of canine pancreatic rough microsomes identifies a novel Hsp40, termed ERj7.

The rough ER (rER) plays a central role in the biogenesis of most extracellular and many organellar proteins in eukaryotic cells. Cells that are specialized in protein secretion, such as pancreatic cells, are particularly rich in rER. In the process of cell homogenization, the rER is converted into ribosome-studded vesicles, the so-called rough microsomes. Here we report on a membrane proteomic analysis of canine pancreatic rough microsomes. Special emphasis was placed on components involved in the various aspects of protein biogenesis, such as protein transport, protein folding, protein modification, and protein degradation. Our results indicate that the Hsp70-chaperone network that is present in the pancreatic ER is even more complex than previously thought, and suggest that the pancreatic rER has a significant capacity for protein degradation.

Characterization of platelet proteins using peptide centric proteomics.

In modern proteomics, undersampling of low abundant, cumbersome, and hydrophobic proteins states one of the major problems. To overcome this, especially in two 2D-PAGE (two-dimensional polyacrylamide gel electrophoresis) eminent drawbacks, the so-called peptide-centric techniques have been developed. These approaches do not separate proteins prior to digestion, but instead proteolytically generate peptide mixtures after it. However, by this procedure already complex protein mixtures become even more extensive peptide mixtures. Particularly, when dealing with large proteomes, the generated sample complexity is vast and therefore difficult to analyze. When separated and analyzed by LC/MS, too many peptides may enter the mass spectrometer at a certain time point, and only a small fraction of ions is selected for subsequent MS/MS analysis. Although protein hydrophobicity and size play minor roles (as long as protease cleavage sites are accessible), low copy number can severely limit identification rates. To reduce the amount of peptides entering the mass spectrometer simultaneously without the loss of overall proteomic information, different techniques have been developed. Among these, an approach is represented by COFRADIC (Combined Fractional Diagonal Chromatography). COFRADIC is a chromatography-based technique enabling the sorting of peptides due to retention time shifts after a specific modification step. In the original approach, a complex peptide mixture is separated by a primary RP-HPLC (reversed-phase high-performance liquid chromatography) run and fractions are retained. Subsequently, these fractions are modified to specifically change retention times of peptides and separated in one or more secondary RP-HPLC runs. In this chapter, COFRADIC approaches for methionine or cysteine containing as well as N-terminal peptides are described. Besides the reduction of sample complexity, the major advantage of COFRADIC might be seen in its versatility. Nearly every feature unique for a subset of peptides, which can be specifically modified by a sorting reaction, is accessible for COFRADIC. Among these are protein phosphorylation, N-glycosylation, and in vivo protein processing sites. Finally, COFRADIC allows the analysis of large numbers of samples and is highly automatable.

Platelet membrane proteomics: a novel repository for functional research.

Being central players in thrombosis and hemostasis, platelets react in manifold and complex ways to extracellular stimuli. Cell-matrix and cell-cell interactions are mandatory for initial adhesion as well as for final development of stable plugs. Primary interfaces for interactions are plasma membrane proteins, of which many have been identified over the past decades in individual studies. However, due to their enucleate structure, platelets are not accessible to large-scale genomic screens and thus a comprehensive inventory of membrane proteins is still missing. For this reason, we here present an advanced proteomic setup for the detailed analysis of enriched platelet plasma membranes and the so far most complete collection of platelet membrane proteins. In summary, 1282 proteins were identified, of which more than half are termed to be of membrane origin. This study provides a brief overview of gene ontology subcellular and functional classification, as well as interaction network analysis. In addition, the mass spectrometric data were used to assemble a first tentative relative quantification of large-scale data on the protein level. We therefore estimate the presented data to be of major interest to the platelet research field and to support rational design of functional studies.

Phosphoproteome of resting human platelets.

Beside their main physiological function in hemostasis, platelets are also highly involved in pathological processes, such as atherothrombosis and inflammation. During hemostasis, binding of adhesive substrates to tyrosine-kinase-linked adhesion receptors and/or soluble agonists to G-protein coupled receptors leads to a cascade of intracellular signaling processes based on substrate (de)phosphorylation. The same mechanisms are involved in platelet activation at sites of atherosclerotic plaque rupture, contributing to vessel occlusion and consequently to pathologic states, such as myocardial infarction, stroke, or peripheral artery disease. To gain a deeper insight into platelet function, we analyzed the phosphoproteome of resting platelets and identified 564 phosphorylation sites from more than 270 proteins, of which many have not been described in platelets before. Among those were several unknown potential protein kinase A (PKA) and protein kinase G (PKG) substrates. Because platelet inhibition is tightly regulated especially by PKA and PKG activity, these proteins may represent important new targets for cardiovascular research. Thus, our finding that GPIbalpha is phosphorylated at Ser603 in resting platelets may represent a novel mechanism for the regulation of one of the most important platelet receptor (GPIb-IX-V) mediated signaling pathways by PKA/PKG.

Silver- and Coomassie-staining protocols: detection limits and compatibility with ESI MS.

Staining protocols for PAGE have to be sensitive and should not impair further MS analysis of selected samples. In this study, the MS compatibility of different silver- and Coomassie-staining protocols with a nano-LC-MS/MS system was systematically elucidated. Altogether, 13 different silver-staining, 1 imidazole-staining and 2 Coomassie-staining protocols were used and compared to each other for their achieved sequence coverage and their detection sensitivity. Three proteins were used as model proteins (bovine serum albumin, rabbit L-lactate dehydrogenase, bovine milk beta-lactoglobulin) in decreasing concentration (12 pmol down to 30 fmol) and different staining protocols were applied. The conclusion of this study is that two silver-staining protocols (Blum, H. et al.,. Electrophoresis 1987, 8, 93-99 and Shevchenko, A. et al.,. Anal. Chem. 1996, 68, 850-858) combine good sequence coverage and good sensitivity and are recommended for nano-LC-MS/MS analysis.

Towards a high resolution separation of human cerebrospinal fluid.

Human cerebrospinal fluid is an ultrafiltrate of plasma that is largely produced by the choroid plexus. It consists of a mixture of anorganic salts, various sugars, lipids and proteins from the surrounding brain tissues. The predominant proteins in cerebrospinal fluid are isoforms of serum albumin, transferrin and immunoglobulins, representing more than 70% of the total protein amount. A rough overview of the protein compounds of human cerebrospinal fluid including their respective concentrations is given by Blennow et al. [Eur. Neurol. 33 (1993) 129]. In contrast, the aim of this work is to display the detailed protein composition of CSF by two-dimensional gel electrophoresis and to identify both high and low concentrated proteins using different mass spectrometry techniques. This extensive overview of proteins in human cerebrospinal fluid will be highly relevant for clinical research. Furthermore, the comparison of 2D gels will help to analyze the standard protein variability in CSF of healthy persons and detect specific protein variations of patients with various neurological diseases (e.g., Alzheimer's disease, Huntington's chorea). Sample preparation for two-dimensional gel electrophoresis must include concentration and desalting steps such as precipitation and ultrafiltration due to the high amount of salts, sugars and lipids and the low total amount of protein of 0.3-0.7 microg/microl present in human CSF. Up to now we were able to identify more than 480 spots from suchlike generated 2D gels using MALDI- and ESI-mass spectrometry.