Oviductal fluid modulates the dynamics of tyrosine phosphorylation in cryopreserved boar spermatozoa during capacitation

Following insemination, spermatozoa are retained in the utero‐tubal junction and isthmic region of the oviduct, where essential steps of capacitation are coordinated. Although a majority of the spermatozoa is exposed to similar conditions in the oviduct, the speed of the response varies depending on the individual male and the state of the spermatozoa. The present study evaluated individual boar variations in terms of the ability of spermatozoa to undergo tyrosine phosphorylation in response to isthmic oviductal fluid (ODF). Cryopreserved spermatozoa from four boars were incubated with pre‐ and post‐ovulatory ODF for 6 hr. Sperm kinematics, global protein tyrosine phosphorylation, and dynamics of different phosphorylation patterns were analyzed at hourly interval. The percentage of phosphorylated spermatozoa in the pre‐ovulatory ODF‐treated group was significantly (P < 0.001) higher than in the other treatment groups. Motility, velocity, and protein tyrosine phosphorylation in spermatozoa in response to ODF and control media also showed differences between boars. Spermatozoa from all four boars showed strong expression of a 19‐kDa phosphoprotein while spermatozoa from two boars showed additionally strong expression of a 32‐kDa phosphoprotein when incubated with pre‐ovulatory ODF. While phosphorylation of proteins in the acrosome and the equatorial segment of the sperm were noticed at an early stage during incubation with ODF, tail phosphorylation appeared at a later stage of capacitation. The results indicate individual variation between boars in terms of sperm proteins, including different phosphorylation patterns, in response to ODF, which might be related to fertility.Mol. Reprod. Dev. 79: 525‐540, 2012. © 2012 Wiley Periodicals, Inc.     

Exploring the oviductal fluid proteome by a lectin‐based affinity approach

The analysis of glycoproteins in body fluids represents a central task in the study of vital processes. Herein, we assessed the combined use of Concanavalin A and Wheat Germ Agglutinin as ligands to fractionate and enrich glycoproteins from oviductal fluid (OF), which is a source of molecules involved in fertilization. First, the selectivity was corroborated by a gel‐based approach using glycoprotein staining and enzymatic deglycosylation. Nanoliquid chromatography‐tandem mass spectrometry (nLC‐ESI‐MS/MS) further allowed the reliable identification of 134 nonbound as well as 130 lectin‐bound OF proteins. Enrichment analysis revealed that 77% of the annotated proteins in the lectin‐bound fraction were known glycoproteins (p‐value [FDR] = 1.45E‐31). The low variance of the number of peptide spectrum matches for each protein within replicates indicated a consistent reproducibility of the whole workflow (median CV 17.3% for technical replicates and 20.7% for biological replicates). Taken together, this study highlights the applicability of a lectin‐based workflow for the comprehensive analysis of OF proteins and gives for the first time an insight into the broad glycoprotein content of OF.     

Proteomic evaluation of wound‐healing processes in potato (Solanum tuberosum L.) tuber tissue

Proteins from potato (Solanum tuberosum L.) tuber slices, related to the wound‐healing process, were separated by 2‐DE and identified by an MS analysis in MS and MS/MS mode. Slicing triggered differentiation processes that lead to changes in metabolism, activation of defence and cell‐wall reinforcement. Proteins related to storage, cell growth and division, cell structure, signal transduction, energy production, disease/defence mechanisms and secondary metabolism were detected. Image analysis of the 2‐DE gels revealed a time‐dependent change in the complexity of the polypeptide patterns. By microscopic observation the polyalyphatic domain of suberin was clearly visible by D4, indicating that a closing layer (primary suberisation) was formed by then. A PCA of the six sampling dates revealed two time phases, D0–D2 and D4–D8, with a border position between D2 and D4. Moreover, a PCA of differentially expressed proteins indicated the existence of a succession of proteomic events leading to wound‐periderm reconstruction. Some late‐expressed proteins (D6–D8), including a suberisation‐associated anionic peroxidase, have also been identified in the native periderm. Despite this, protein patterns of D8 slices and native periderm were still different, suggesting that the processes of wound‐periderm formation are extended in time and not fully equivalent. The information presented in this study gives clues for further work on wound healing‐periderm formation processes.     

Targeted serum glycoproteomics for the discovery of lung cancer‐associated glycosylation disorders using lectin‐coupled ProteinChip arrays

To screen for glycoproteins showing aberrant sialylation patterns in sera of cancer patients and apply such information for biomarker identification, we performed SELDI‐TOF MS analysis coupled with lectin‐coupled ProteinChip arrays (Jacalin or SNA) using sera obtained from lung cancer patients and control individuals. Our approach consisted of three processes (i) removal of 14 abundant proteins in serum, (ii) enrichment of glycoproteins with lectin‐coupled ProteinChip arrays, and (iii) SELDI‐TOF MS analysis with acidic glycoprotein‐compatible matrix. We identified 41 protein peaks showing significant differences (p<0.05) in the peak levels between the cancer and control groups using the Jacalin‐ and SNA‐ProteinChips. Among them, we identified loss of Neu5Ac (α2,6) Gal/GalNAc structure in apolipoprotein C‐III (apoC‐III) in cancer patients through subsequent MALDI‐QIT‐TOF MS/MS. Furthermore, subsequent validation experiments using an additional set of 60 lung adenocarcinoma patients and 30 normal controls demonstrated that there is a higher frequency of serum apoC‐III with loss of α2,6‐linkage Neu5Ac residues in lung cancer patients compared to controls. Our results have demonstrated that lectin‐coupled ProteinChip technology allows the high‐throughput and specific recognition of cancer‐associated aberrant glycosylations, and implied a possibility of its applicability to studies on other diseases.     

Identification,Quantification, and System Analysis of Protein N‐ε Lysine Methylation in Anucleate Blood Platelets

Protein posttranslational modifications critically regulate a range of physiological and disease processes. In addition to tyrosine, serine, and threonine phosphorylation, reversible N‐ε acylation and alkylation of protein lysine residues also modulate diverse aspects of cellular function. Studies of lysine acyl and alkyl modifications have focused on nuclear proteins in epigenetic regulation; however, lysine modifications are also prevalent on cytosolic proteins to serve increasingly apparent, although less understood roles in cell regulation. Here, the methyl‐lysine (meK) proteome of anucleate blood platelets is characterized. With high‐resolution, multiplex MS methods, 190 mono‐, di‐, and tri‐meK modifications are identified on 150 different platelet proteins—including 28 meK modifications quantified by tandem mass tag (TMT) labeling. In addition to identifying meK modifications on calmodulin (CaM), GRP78 (HSPA5, BiP), and EF1A1 that have been previously characterized in other cell types, more novel modifications are also uncovered on cofilin, drebin‐like protein (DBNL, Hip‐55), DOCK8, TRIM25, and numerous other cytoplasmic proteins. Together, the results and analyses support roles for lysine methylation in mediating cytoskeletal, translational, secretory, and other cellular processes. MS data for this study have been deposited into the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD012217.     

Characterization of the Proteome of Theobroma cacao Beans by Nano‐UHPLC‐ESI MS/MS

Cocoa seed storage proteins play an important role in flavour development as aroma precursors are formed from their degradation during fermentation. Major proteins in the beans of Theobroma cacao are the storage proteins belonging to the vicilin and albumin classes. Although both these classes of proteins have been extensively characterized, there is still limited information on the expression and abundance of other proteins present in cocoa beans. This work is the first attempt to characterize the whole cocoa bean proteome by nano‐UHPLC‐ESI MS/MS analysis using tryptic digests of cocoa bean protein extracts. The results of this analysis show that >1000 proteins could be identified using a species‐specific Theobroma cacao database. The majority of the identified proteins were involved with metabolism and energy. Additionally, a significant number of the identified proteins were linked to protein synthesis and processing. Several proteins were also involved with plant response to stress conditions and defence. Albumin and vicilin storage proteins showed the highest intensity values among all detected proteins, although only seven entries were identified as storage proteins. A comparison of MS/MS data searches carried out against larger non‐specific databases confirmed that using a species‐specific database can increase the number of identified proteins, and at the same time reduce the number of false positives. The results of this work will be useful in developing tools that can allow the comparison of the proteomic profile of cocoa beans from different genotypes and geographic origins. Data are available via ProteomeXchange with identifier PXD005586.     

An Integrated Proteomic Approach Uncovers Novel Substrates and Functions of the Lon Protease in Escherichia coli

Controlling the cellular abundance and proper function of proteins by proteolysis is a universal process in all living organisms. In Escherichia coli, the ATP‐dependent Lon protease is crucial for protein quality control and regulatory processes. To understand how diverse substrates are selected and degraded, unbiased global approaches are needed. We employed a quantitative Super‐SILAC (stable isotope labeling with amino acids in cell culture) mass spectrometry approach and compared the proteomes of a lon mutant and a strain producing the protease to discover Lon‐dependent physiological functions. To identify Lon substrates, we took advantage of a Lon trapping variant, which is able to translocate substrates but unable to degrade them. Lon‐associated proteins were identified by label‐free LC‐MS/MS. The combination of both approaches revealed a total of 14 novel Lon substrates. Besides the identification of known pathways affected by Lon, for example, the superoxide stress response, our cumulative data suggests previously unrecognized fundamental functions of Lon in sulfur assimilation, nucleotide biosynthesis, amino acid and central energy metabolism.     

Comparison of sialylated N‐glycopeptide levels in serum of pancreatic cancer patients,acute pancreatitis patients,and healthy controls

Serum protein glycosylation is known to be affected by pathological conditions, including cancer and inflammatory diseases. Pancreatic cancer patients would benefit from early diagnosis, as the disease is often detected in an advanced stage and has poor prognosis. Searching for changes in serum protein site‐specific glycosylation could reveal novel glycoprotein biomarkers. We used Sambucus nigra lectin affinity chromatography to enrich α‐2,6 sialylated tryptic N‐glycopeptides from albumin‐depleted sera of pancreatic cancer patients, acute pancreatitis patients, and healthy individuals, and compared their relative abundance using ultra performance LC‐MS. Relative quantitation was done using the spectrum processing software MZmine. Identification was performed on the web‐based tool GlycopeptideID, developed for in silico analysis of intact N‐glycopeptides. Seventeen high‐abundance serum proteins, mainly acute‐phase proteins, and immunoglobulins, with total 27 N‐glycosylation sites, and 62 glycoforms, were identified. Pancreatitis patient sera contained 38, and pancreatic cancer patients sera contained 13 glycoform changes with statistical significance (p < 0.05). In pancreatitis, up to tenfold changes were found in some glycoforms, and in pancreatic cancer, threefold. Analysis showed that the changes often concerned one or two, but not all, N‐glycosylation sites in a specific glycoprotein. In conclusion, the analysis shows that pancreatic cancer, and acute pancreatitis are associated with changes in concentrations of intact sialylated N‐glycopeptides derived from acute‐phase proteins, and immunoglobulins, and that changes are site specific.     

Identification of Neospora caninum proteins regulated during the differentiation process from tachyzoite to bradyzoite stage by DIGE

Identification of differentially expressed proteins during Neospora caninum tachyzoite–bradyzoite conversion processes may lead to a better knowledge of the pathogenic mechanisms developed by this important parasite of cattle. In the present work, a differential expression proteomic study of tachyzoite and bradyzoite stages was accomplished for the first time by applying DIGE technology coupled with MS analysis. Up to 72 differentially expressed spots were visualized (1.5‐fold in relative abundance, p<0.05, t‐test). A total of 53 spots were more abundant in bradyzoites and 19 spots in tachyzoites. MS analysis identified 26 proteins; 20 of them overexpressed in the bradyzoite stage and 6 in the tachyzoite stage. Among the novel proteins, enolase and glyceraldehyde‐3‐phosphate dehydrogenase (involved in glycolysis), HSP70 and HSP90 (related to stress response) as well as the dense granule protein GRA9, which showed higher abundance in the bradyzoite stage, might be highlighted. On the other hand, isocitrate dehydrogenase 2, involved in the Krebs cycle, was found to be more abundant in tachyzoites extract. Biological functions from most novel proteins were correlated with previously reported processes during the differentiation process in Toxoplasma gondii. Thus, DIGE technology arises as a suitable tool to study mechanisms involved in the N. caninum tachyzoite to bradyzoite conversion.     

Developing a strong anion exchange/RP (SAX/RP) 2D LC system for high‐abundance proteins depletion in human plasma

Human plasma is dominated by high‐abundance proteins which severely impede the detection of low‐abundance proteins. Unfortunately, now there is no efficient method for large‐scale depletion of high‐abundance proteins in human plasma. In this study, we developed a new strategy, strong anion exchange (SAX)/RP 2D LC system, which has potential for large‐scale depletion of high‐abundance proteins in human plasma. Separation gradients of the system were optimized to ensure an extensive separation of plasma proteins. Plasma was fractionated into 67 fractions by SAX. All these fractions were subjected a thorough separation by the 2D RPLC and 66 peaks with high UV absorption (>20 mAU) at 215 nm were collected. Proteins in these peaks were identified by LC‐MS/MS analysis. Results showed that 83 proteins could be identified in these peaks, 68 among them were reported to be high‐ or middle‐abundance proteins in plasma. All these proteins had definite retention times and were mapped in the 2D SAX‐RP system, which resulted in accurate depletion of high‐abundance proteins with ease. Our studies provide a convenient and effective method for large‐scale depletion of high‐abundance proteins and in‐depth research in human plasma proteomics.     

Mass Spectrometry‐Based Analysis of Time‐Resolved Proteome Quantification

The aspect of time is essential in biological processes and thus it is important to be able to monitor signaling molecules through time. Proteins are key players in cellular signaling and they respond to many stimuli and change their expression in many time‐dependent processes. Mass spectrometry (MS) is an important tool for studying proteins, including their posttranslational modifications and their interaction partners—both in qualitative and quantitative ways. In order to distinguish the different trends over time, proteins, modification sites, and interacting proteins must be compared between different time points, and therefore relative quantification is preferred. In this review, the progress and challenges for MS‐based analysis of time‐resolved proteome dynamics are discussed. Further, aspects on model systems, technologies, sampling frequencies, and presentation of the dynamic data are discussed.     

Adsorption of oviductal fluid proteins by the bovine sperm membrane during in vitro capacitation.

125I-labeled oviductal fluid (ODF) proteins and antiserum to ODF were used to determine whether ODF proteins associate with the sperm membrane during in vitro capacitation. Luteal and nonluteal pools of ODF were obtained from oviduct catheters during the estrous cycle. Washed sperm (50 x 10(6) sperm/ml) were incubated up to 4 h in a protein-free modified Tyrode's medium (MTM), or MTM supplemented with 40% ODF, or 0.5 ng 125I-labeled ODF proteins. Solubilized sperm membrane proteins and incubation media containing ODF proteins were separated by gel electrophoresis. Membranes isolated from bovine sperm, previously incubated with ODF, adsorbed five 125I-proteins: A doublet at 85-95 kDa, and others at 24, 34, 53, and 66 kDa. The amount of 66 kDA 125I-protein associated with the sperm decreased during the incubation, whereas the amount of 85 to 95-kDa protein did not. Western blot analyses also detected the presence of ODF proteins (53, 66, 85-95, and 116 kDa) in solubilized membranes from sperm incubated in ODF. The 85 to 95-kDa protein in ODF decreased in apparent molecular weight by 5 kDa when associated with the sperm membrane. At 53 kDa, ODF proteins which associated with sperm were transformed from two to three separate proteins. These studies indicate that the surface of sperm is modified by adsorption of ODF proteins to the membrane during in vitro capacitation.     

Design of five‐layer gold nanoparticles self‐assembled in a liquid open tubular column for ultrasensitive nano‐LC‐MS/MS proteomic analysis of 80 living cells

In this work, for the first time, a liquid open tubular column modified by five‐layer gold nanoparticles and linked with C₁₈ (GNPs@C₁₈) was designed and fabricated for nano‐LC‐MS/MS analysis of 80 living cells. Sixty nanometer gold nanoparticles were self‐assembled layer by layer on the inner wall of a 20 μm id fused‐silica capillary. C₁₈ was then linked on the gold nanoparticles to make the liquid open tubular column show hydrophobic character. Enough loading capacities for analysis of 80 living cells, ～100 fmol for pk‐10 and ～30 fmol for insulin, were obtained with the 2 m × 20 μm id five‐layer GNPs@C₁₈ open tubular column. The open tubular column was used in an online pretreatment and direct nano‐LC‐MS/MS analysis system to analyze 80 living HepG2 cells. In total, 650 proteins were identified in triplicate runs. The subcellular localization of the identified proteins showed that our system had no bias toward different cellular compartments. Protein copy number per cell of the identified proteins showed that the detection limit could reach 50 zmol and the abundance of the identified proteins could cover a dynamic range of 6 orders.     

SILAC‐based quantitative proteomic analysis of secretome of Marc‐145 cells infected with porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of PRRS, which causes severe reproductive failure in sows, respiratory disease in young and growing pigs, and enormous economic losses to the global swine industry. In this study, SILAC combined with MS/MS was used to quantitatively identify the secretory proteins differentially expressed in PRRSV‐infected Marc‐145 cells compared with mock‐infected controls. In total, we identified 204 secretory proteins showing significant differences in infected cells (163 upregulated, 41 downregulated). Intensive bioinformatic analysis of secretome data revealed that PRRSV infection strongly activated nonclassical protein secretion, especially vesicle‐mediated release of exosomal proteins, including different danger‐associated molecular pattern molecules and the majority of secreted proteins involved in protein binding and transport, regulation of response to stimulus, metabolic processes, and immune responses. According to the functional proteins analysis, we speculate that proteins functioning in binding, transport, and the immune response are exploited by PRRSV to facilitate virus replication and immune evasion. Our study for the first time analyzes the secretory protein profile of PRRSV‐infected Marc‐145 cells and provides valuable insight into the host response to PRRSV infection.     

Proteomics analysis of adult testis from Bombyx mori

The development of the testis involves a large number of tissue‐specific proteins, possibly because the sperms in it are the most divergent of all cell types. In this study, LC‐MS/MS was employed to investigate the protein compositions of the adult testis of silkworm. A total of 14 431 peptides were identified in the adult testis of Bombyx mori, which were matched to 2292 proteins. Thirty‐two HSPs constitute a group of most abundant proteins in the adult testis, suggesting that they are critical for the development, differentiation, and survival of germ cells. Other proteins in this analysis were also involved in testis‐specific processes mainly including sperm motility, meiosis, germ cell development, and spermatogenesis. The data have been deposited to the ProteomeXchange with identifier PXD000909 ( http://proteomecentral.proteomexchange.org/dataset/PXD000909 ).     

Monosaccharides are not detected in whole or isthmic bovine oviductal fluid collected throughout the estrous cycle, as analyzed by HPLC

In the bovine oviduct, monosaccharides may play a role in the preparation of gametes for fertilization. Sperm are sequestered in the isthmic region of the oviduct where capacitation, requisite biochemical changes in sperm membranes, may take place. Retention of spermatozoa in the oviductal isthmus is dependent on a carbohydrate recognition system between oviductal epithelium and sperm membrane lectins. The monosaccharide, fucose, has been found to be important to this recognition system. However, both gametes and epithelium are also bathed in oviductal fluid (ODF), and fucose or other monosaccharides may be constituents of ODF and so may be important to sperm binding to oviductal epithelium and subsequent preparation for fertilization. In this study, ODF from dairy cows was analyzed by HPLC for the presence of 5 monosaccharides (fucose, galactose, glucosamine, mannose and xylose). Both whole ODF, collected by cannulation of the entire oviduct of 1 cow over a complete estrous cycle, and regional staged ODF, collected and pooled from 13 cows from the isthmic region only at estrus, were analyzed. We report negligible concentrations of all 5 monosaccharides in both types of ODF analyzed. Because the detection limit of our assay was 10(8) times lower than fucose concentrations found to be physiologically important in earlier in vitro studies, we conclude that bovine ODF does not contain physiologically active levels of free fucose or other, similar monosaccharides at any time of the estrous cycle.