A systematic characterization of mitochondrial proteome from human T leukemia cells

Global understanding of tissue-specific differences in mitochondrial signal transduction requires comprehensive mitochondrial protein identification from multiple cell and tissue types. Here, we explore the feasibility and efficiency of protein identification using the one-dimensional gel electrophoresis in combination with the nano liquid-chromatography tandem mass spectrometry (GeLC-MS/MS). The use of only 40 mug of purified mitochondrial proteins and data analysis using stringent scoring criteria and the molecular mass validation of the gel slices enables the identification of 227 known mitochondrial proteins (membrane and soluble) and 453 additional proteins likely to be associated with mitochondria. Replicate analyses of 60 mug of mitochondrial proteins on the faster scanning LTQ mass spectrometer validate all the previously identified proteins and most of the single hit proteins except the 81 single hit proteins. Among the identified proteins, 466 proteins are known to functionally participate in various processes such as respiration, tricarboxylic acid cycle (TCA cycle), amino acid and nucleotide metabolism, glycolysis, protection against oxidative stress, mitochondrial assembly, molecular transport, protein biosynthesis, cell cycle control, and many known cellular processes. The distribution of identified proteins in terms of size, pI, and hydrophobicity reveal that the present analytical strategy is largely unbiased and very efficient. Thus, we conclude that this approach is suitable for characterizing subcellular proteomes form multiple cells and tissues.     

Assessment of some tools for the characterization of the human osteoarthritic cartilage proteome.

Since the proteome of osteoarthritic articular cartilage has been poorly investigated as yet, we adapted proteomic technologies to the study of the proteins secreted or released by fresh human osteoarthritic cartilage in culture. Fresh cartilage explants were obtained from three donors undergoing surgery for knee joint replacement. The explants were dissected out, minced, and incubated in serum-free culture medium. After 48 h, proteins in the medium were identified by two-dimensional or off-gel electrophoresis coupled to tandem mass spectrometry, or by using an antibody-based protein microarray designed to detect angiogenic factors, growth factors, chemokines, and cytokines. We identified a series of 43 proteins. Some of these proteins were already described as secretion products of chondrocytes, such as YKL-39 or osteoprotegerin, while several other were known proteins but have never been reported previously in cartilage, such as the serum amyloid P-component, the vitamin D binding protein, the pigment epithelium derived factor, the pulmonary and activation-regulated chemokine, lyl-1, thrombopoietin, fibrinogen, angiogenin, gelsolin, and osteoglycin/mimecan. While this study enabled the identification of novel proteins secreted or released by human osteoarthritic cartilage, the goal of the present work was essentially to describe the technical approach necessary for a systematic study of osteoarthritic cartilages from a large population of donors, in order to be able to select the good markers and/or targets for this poorly explored disease.     

Aggregation propensity of the human proteome

Formation of amyloid-like fibrils is involved in numerous human protein deposition diseases, but is also an intrinsic property of polypeptide chains in general. Progress achieved recently now allows the aggregation propensity of proteins to be analyzed over large scales. In this work we used a previously developed predictive algorithm to analyze the propensity of the 34,180 protein sequences of the human proteome to form amyloid-like fibrils. We show that long proteins have, on average, less intense aggregation peaks than short ones. Human proteins involved in protein deposition diseases do not differ extensively from the rest of the proteome, further demonstrating the generality of protein aggregation. We were also able to reproduce some of the results obtained with other algorithms, demonstrating that they do not depend on the type of computational tool employed. For example, proteins with different subcellular localizations were found to have different aggregation propensities, in relation to the various efficiencies of quality control mechanisms. Membrane proteins, intrinsically disordered proteins, and folded proteins were confirmed to have very different aggregation propensities, as a consequence of their different structures and cellular microenvironments. In addition, gatekeeper residues at strategic positions of the sequences were found to protect human proteins from aggregation. The results of these comparative analyses highlight the existence of intimate links between the propensity of proteins to form aggregates with β-structure and their biology. In particular, they emphasize the existence of a negative selection pressure that finely modulates protein sequences in order to adapt their aggregation propensity to their biological context.     

Analysis of the human saliva proteome

Interest in the characterization of the salivary proteome has increased in the last few years. This review discusses the different techniques and methodologies applied to the separation and identification of salivary proteins. Nowadays, proteomic techniques are the state of the art for the analysis of biologic materials and saliva is no exception. 2D electrophoresis and tryptic digest analysis by mass spectrometry are the typical methodology, but new approaches using 2D liquid chromatography/mass spectrometry methods have already been introduced for saliva analysis. Due to their important physiologic role in the oral cavity, low-molecular-weight proteins and peptides are also included in this article and the methodologies discussed.     

Variability of the healthy human proteome

The aim of this review is to analyze results of studies on characteristics of protein variability and diversity of posttranslational modifications of proteins in healthy humans. Numerous studies have demonstrated that a proteomic profile is characterized by significant intra- and inter-individual variability, and quite often natural (“normal”) variability of some proteins can be comparable to changes observed in pathological processes. Results obtained by our research group have demonstrated high intra-individual variability of serum low-molecular subproteome of healthy volunteers, certified by a special medial committee (the coefficient of variation (CV) of 42.6%). The proteins characterized by high variability under normal conditions (e.g. haptoglobin — 0–40 mg/ml; lysozyme — 0.01–0.1 mg/ml; C-reactive protein — 0.01–0.3 mg/ml) cannot be considered as potential biomarkers of diseases. On the contrary, proteins and peptides characterized by insignificant dispersion in healthy population (such as albumin ( CV = 9%); transferrin-(CV = 14%); C3c complement (CV = 17%), α-1 acid glycoprotein (CV = 21%), α-2-macroglobulin (CV = 20%); transthyretin fragment (CV = 28.3%) and α2-HS-glycoprotein βchain (CV = 29.7%)) can provide valuable information about the state of health. Thus, studies of plasticity in the proteomic profiles of healthy humans will help to correct reference intervals used in clinical proteomics.     

Analysis of the human saliva proteome

Interest in the characterization of the salivary proteome has increased in the last few years. This review discusses the different techniques and methodologies applied to the separation and identification of salivary proteins. Nowadays, proteomic techniques are the state of the art for the analysis of biologic materials and saliva is no exception. 2D electrophoresis and tryptic digest analysis by mass spectrometry are the typical methodology, but new approaches using 2D liquid chromatography/mass spectrometry methods have already been introduced for saliva analysis. Due to their important physiologic role in the oral cavity, low-molecular-weight proteins and peptides are also included in this article and the methodologies discussed.     

Analysis of the human serum proteome

Changes in serum proteins that signal histopathological states, such as cancer, are useful diagnostic and prognostic biomarkers. Unfortunately, the large dynamic concentration range of proteins in serum makes it a challenging proteome to effectively characterize. Typically, methods to deplete highly abundant proteins to decrease this dynamic protein concentration range are employed, yet such depletion results in removal of important low abundant proteins. A multi-dimensional peptide separation strategy utilizing conventional separation techniques combined with tandem mass spectrometry (MS/MS) was employed for a proteome analysis of human serum. Serum proteins were digested with trypsin and resolved into 20 fractions by ampholyte-free liquid phase isoelectric focusing. These 20 peptide fractions were further fractionated by strong cation-exchange chromatography, each of which was analyzed by microcapillary reversed-phase liquid chromatography coupled online with MS/MS analysis. This investigation resulted in the identification of 1444 unique proteins in serum. Proteins from all functional classes, cellular localization, and abundance levels were identified. This study illustrates that a majority of lower abundance proteins identified in serum are present as secreted or shed species by cells as a result of signalling, necrosis, apoptosis, and hemolysis. These findings show that the protein content of serum is quite reflective of the overall profile of the human organism and a conventional multidimensional fractionation strategy combined with MS/MS is entirely capable of characterizing a significant fraction of the serum proteome. We have constructed a publicly available human serum proteomic database (http://bpp.nci.nih.gov) to provide a reference resource to facilitate future investigations of the vast archive of pathophysiological content in serum. These authors contributed equally to this work.     

Establishment of a reliable method for direct proteome characterization of human articular cartilage

Articular cartilage consists mainly of extracellular matrix, mostly made of collagens and proteoglycans. These macromolecules have so far impaired the detailed two-dimensional electrophoresis-based proteomic analysis of articular cartilage. Here we describe a method for selective protein extraction from cartilage, which excludes proteoglycans and collagen species, thus allowing direct profiling of the protein content of cartilage by two-dimensional electrophoresis. Consistent electrophoretic patterns of more than 600 protein states were reproducibly obtained after silver staining from 500 mg of human articular cartilage from joints with diverse pathologies. The extraction yield increased when the method was applied to a chondrosarcoma sample, consistent with selective extraction of cellular components. Nearly 200 of the most intensely stained protein spots were analyzed by MALDI-TOF mass spectrometry after trypsin digestion. They represented 127 different proteins with diverse functions. Our method provides a rapid, efficient, and pertinent alternative to previously proposed approaches for proteomic characterization of cartilage phenotypes. It will be useful for detecting protein expression patterns that relate pathophysiological processes of cartilaginous tissues such as osteoarthritis and chondrosarcoma.     

Partial characterization of the proteome of the mouse striatum

Many diseases of the mammalian CNS, including Parkinson's (PD) and Lesch Nyhan disease (LND), are associated with programmatic neurodegeneration or dysfunction of dopaminergic neurons in the mesencephalon, the nigrostriatal pathway, and its projections in the striatum [1-4]. Proteomic studies on brain tissue of both animal models and human PD patients have provided evidence for dysfunction and damage of many pathways, including oxidative stress-related damage, ubiquitin-proteasome dysfunction, mitochondrial energy metabolism deficiencies, and synaptic function [5-11]. To date no such proteomic studies have been reported in the related and rare basal ganglia disorder LND, a developmental rather than a neurodegenerative neurological disorder caused by deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) that regulates a major step in the purine salvage pathway [12]. Many studies have demonstrated that the both human LND patients and a mouse knockout model of HPRT deficiency have significantly reduced levels and uptake of dopamine in the striatum [4, 13-16] that is likely to be the principal cause of the CNS disorder. The precise molecular and cellular mechanisms that underlie this neurotransmitter defect are unknown.     

Initial proteome analysis of mature barley seeds and malt

Several barley (Hordeum vulgare) cultivars are used in the production of malt for brewing. The malt quality depends on the cultivar, its growth and storage conditions, and the industrial process. To enhance studies on malt quality, we embarked on a proteome analysis approach for barley seeds and malt. The proteome analysis includes two-dimensional (2-D) gel electrophoresis, mass spectrometry, and bioinformatics for identification of selected proteins. This project initially focused on proteins in major spots in the neutral isoelectric point range (pI 4-7) including selected spots that differ between four barley cultivars. The excellent malting barley cultivar Barke was used as reference. Cultivar differences in the 2-D gel spot patterns are observed both at the seed and the malt level. In seed extracts one of the proteins causing variations has been identified as an alpha-amylase/trypsin inhibitor. In malt extracts multiple forms of the alpha-amylase isozyme 2 have been identified in varying cultivar characteristic spot patterns. The present identification of proteins in major spots from 2-D gels includes 27 different proteins from 42 spots from mature seed extract, while only three specific proteins were identified by analysing 13 different spots from the corresponding malt extract. It is suggested that post-translational processing causes the same protein to occur in different spots.     

The human oligodendrocyte proteome

Myelination of the CNS is performed by oligodendrocytes (OLs), which have been implicated in brain disorders, such as multiple sclerosis and schizophrenia. We have used the human oligodendroglial cell line MO3.13 to establish an OL reference proteome database. Proteins were prefractionationated by SDS‐PAGE and after in‐gel digestion subjected to nanoflow LC‐MS analysis. Approximately 11 600 unique peptides were identified and, after stringent filtering, resulted in 2290 proteins representing nine distinct biological processes and various molecular classes and functions. OL‐specific proteins, such as myelin basic protein (MBP) and 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNP), as well as other proteins involved in multiple sclerosis and schizophrenia were also identified and are discussed. Proteins of this dataset have also been classified according to their chromosomal origin for providing useful data to the Chromosome‐centric Human Proteome Project (C‐HPP). Given the importance of OLs in the etiology of demyelinating and oligodendrogial disorders, the MO3.13 proteome database is a valuable data resource. The MS proteomics data have been deposited to the ProteomeXchange with identifier PXD000263 ( http://proteomecentral.proteomexchange.org/dataset/PXD000263 ).     

In-depth analysis of the human tear proteome

The tears, a critical body fluid of the surface of the eye, contain an unknown number of molecules including proteins/peptides, lipids, small molecule metabolites, and electrolytes. There have been continued efforts for exploring the human tear proteome to develop biomarkers of disease. In this study, we used the high speed TripleTOF 5600 system as the platform to analyze the human tear proteome from healthy subjects (3 females and 1 male, average age: 36±14). We have identified 1543 proteins in the tears with less than 1% false discovery rate, which represents the largest number of human tear proteins reported to date. The data set was analyzed for gene ontology (GO) and compared with the human plasma proteome, NEIBank lacrimal gland gene dataset and NEIBank cornea gene dataset. This comprehensive tear protein list may serve as a reference list of human tear proteome for biomarker research of ocular diseases or establishment of MRM (Multiple Reaction Monitoring) assays for targeted analysis. Tear fluid is a useful and an accessible source not only for evaluating ocular surface tissues (cornea and conjunctiva), inflammation, lacrimal gland function and a number of disease conditions, such as dry eye as well as response to treatment.     

Characterization of the human blood plasma proteome

We describe methods for broad characterization of the human plasma proteome. The combination of stepwise immunoglobulin G (IgG) and albumin protein depletion by affinity chromatography and ultrahigh-efficiency capillary liquid chromatography separations coupled to ion trap-tandem mass spectrometry enabled identification of 2392 proteins from a single plasma sample with an estimated confidence level of > 94%, and an additional 2198 proteins with an estimated confidence level of 80%. The relative abundances of the identified proteins span a range of over eight orders of magnitude in concentration (< 30 pg/mL to approximately 30 mg/mL), facilitated by the attomole-level sensitivity of the analysis methods. More than 80% of the observed proteins demonstrate interactions with IgG and/or albumin, and the human plasma protein loss in the affinity chromatography/strong cation exchange/reversed-phase liquid chromatography-tandem mass spectrometry methodology was investigated in detail. The results of this study provide a basis for a wide range of plasma proteomics studies, including broad quantitation of relative abundances in comparative studies of the identification of novel protein disease markers, as well as further studies of protein-protein interactions.     

Current knowledge of the human sperm proteome

The knowledge of the mature sperm proteome is undoubtedly the basis for understanding sperm function, the mechanisms responsible for fertilization, the reasons for infertility and possible treatments. The methods of sperm protein extraction depend mainly on the proteins of interest and the protein separation techniques that will be employed. The isolation of the membrane proteins appears to be most problematic step. Nevertheless, two-dimensional electrophoresis and mass spectrometry have become the main techniques used in human sperm protein analysis. We outline the present techniques used to examine the sperm proteome and data generated from studies on the human sperm and different types of male infertility. We present the most characteristic proteins that are involved in sperm function. Their value as biomarkers for diagnosis and treatment of infertility would require further validation. We focus on selected and critical studies of the human sperm proteome to present our subjective view of this fast-moving field.