Proteomic analysis of a membrane skeleton fraction from human liver

The cytoskeleton networks around liver cell cortex can resist Triton extraction and co-pellet with their tightly associated integral membrane proteins, forming assemblies called "membrane skeletons". Despite their important roles in determining cell shape and in signal transduction pathways, the membrane skeletons of human liver cells are uncharacterized to a great extent. In the present work, we prepared a membrane skeleton fraction by Triton extraction of human liver plasma membranes and then separated its protein components by 2-D gels. We optimized the detergent used for protein solubilization and found that 2% ASB-14 allowed the best recovery of membrane skeleton proteins. By analyzing the protein spots with MALDI-TOF and MALDI-TOF-TOF MS, we identified 104 nonredundant proteins, wherein 38 were cytoskeletal proteins that were further classified into several groups, including proteins in fodrin-based meshworks, adhesion proteins (proteins involved in adherens junctions, focal adhesions, desmosomes, hemidesmosomes and tight junctions), proteins that regulate F-actin dynamics, motor proteins, and some other cytoskeletal proteins. To the best of our knowledge, this is one of the largest data sets of membrane skeleton proteins to date. All the results suggested that the liver cells had complex actin- and cytokeratin-based membrane skeletons. This work provided a representative 2-DE map of membrane skeletons from human normal liver, for the purpose of helping to elucidate the composition and function of the membrane skeletons.     

Proteomic analysis of the hydrophobic fraction of mesenchymal stem cells derived from human umbilical cord blood

Mesenchymal stem cells (MSCs) are promising candidates for cell therapy and tissue engineering, but their application has been impeded by lack of knowledge of their core biological properties. In order to identify MSC-specific proteins, the hydrophobic protein fraction was individually prepared from two different umbilical cord blood (UCB)-derived MSC populations; these were then subjected to two-dimensional (2D) gel electrophoresis and peptide mass fingerprinting matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF)-mass spectrometry (MS). Although the 2D gel patterns differed somewhat between the two samples, computer-assisted image analysis identified shared protein spots. 35 spots were reliably identified corresponding to 32 different proteins, many of which were chaperones. Based on their primary sub-cellular locations the proteins could be grouped into 6 categories: extracellular, cell surface, endoplasmic reticular, mitochondrial, cytoplasmic and cytoskeletal proteins. This map of the water-insoluble proteome may provide valuable insights into the biology of the cell surface and other compartments of human MSCs.     

Proteomic analysis of human tears: defensin expression after ocular surface surgery

Human tear protein profiles were monitored by surface enhanced laser desorption/ionization-time-of-flight mass spectrometry ProteinChip technology (SELDI-TOF ProteinChip) and liquid chromatography-mass spectrometry (LC-MS). Tears were collected from 21 patients scheduled for surgery to remove an ocular surface neoplasm prior to surgery (day 0) and on days 1, 3, and 30 postoperatively. Using this proteomic approach, we verified that three human alpha-defensins (HNP-1, HNP-2, and HNP-3) were significantly up-regulated in their expression after surgery and that their levels decreased to approximately normal by day 30 by which time healing was complete. Further confirmation of the identity of the alpha-defensins in human tears was made by LC purification, trypsin digestion, and ESI-MS/MS analysis of their tryptic digests. The concentrations of HNP-1 and HNP-2 were determined and shown to be markedly increased after ocular surface surgery. The results of the study suggest that human alpha-defensins HNP-1, HNP-2, and HNP-3 are up-regulated after surgery, and may in addition to their antimicrobial properties have an important role in wound healing.     

Proteomic expression analysis of human colorectal cancer: Identification of soluble overexpressed proteins

Colorectal cancer is one of the most common malignancies in developed countries. Scarce clinical signs at the early stages of the disease and the lack of fast and sensitive diagnostic techniques based on the detection of tumor specific protein markers contribute greatly to the high mortality rate. The search for such markers is significantly complicated by the high levels of major structural and cytoskeletal proteins in normal and tumor tissues. Extraction with 0.2 M NaCl in the presence of the nonionic detergent NP-40 was performed to enrich the soluble protein fraction. This modification resulted in a considerably increased sensitivity of detection of minor proteins that may enter the circulation during carcinogenesis. The soluble protein profiles of the paired colon adenocarcinoma and normal tissue specimens were compared using 2D gel electrophoresis, which enabled the detection of 10 proteins whose levels in tumors were elevated at least 10-fold as compared to normal tissue. The proteins were identified by MALDI-TOF mass spectrometry, and two new protein markers of colon cancer, TAF9 and CISH, were discovered. Low levels of CISH synthesis in most normal human tissues and tumors other than colorectal cancer make it a prospective candidate diagnostic marker for this type of cancer.     

Proteomic analysis of the cell envelope fraction of Escherichia coli

We applied proteomics technologies to analyze a membrane preparation of Escherichia coli, wild type strain and of transformants expressing human cytochrome P450s. The proteins were analyzed by two-dimensional electrophoresis and identified by matrix-assisted laser desorption ionization mass spectrometry. The membrane proteins were solubilized with both mild detergents such as CHAPS and strong detergents, such as sodium and lithium dodecyl sulfate, sodium cholate and sodium deoxycholate. In the E. colimembrane fraction, 394 different gene products were identified. Approximately 28% of them were predicted to be integral membrane proteins, of which 100 proteins have been predicted to carry one transmembrane region, ten proteins to carry two, and two proteins to include three transmembrane domains. The remaining are probably membrane-associated and cytosolic proteins. Cytochrome P450s did not enter the immobilized pH gradient strips but were efficiently analyzed in a two-dimensional, two-detergent system. Use of strong solubilizing agents resulted in the detection of about 20 membrane proteins, which were not detected following extraction with mild detergents and chaotropes. The present database is one of the largest for membrane proteins.     

Purification and characterization of leucine-specific aminopeptidase from the soluble fraction of human placenta

A soluble aminopeptidase distinct from two enzymes described previously was isolated from human placenta and some of its properties were investigated. The three aminopeptidases were separated by DEAE-cellulose chromatography. The newly found aminopeptidase exhibits specific hydrolysis of leucine derivatives among various synthetic substrates. However, a broad substrate specificity was observed toward some natural bioactive peptides.     

Proteomic DIGE analysis of the mitochondria‐enriched fraction from aged rat skeletal muscle

Skeletal muscle aging is associated with a loss in tissue mass and contractile strength, as well as fiber type shifting and bioenergetic adaptation processes. Since mitochondria represent the primary site for energy generation via oxidative phosphorylation, we investigated potential changes in the expression pattern of the mitochondrial proteome using the highly sensitive DIGE approach. The comparative analysis of the mitochondria‐enriched fraction from young adult versus aged muscle revealed an age‐related change in abundance for 39 protein species. MS technology identified the majority of altered proteins as constituents of muscle mitochondria. An age‐dependent increase was observed for NADH dehydrogenase, the mitochondrial inner membrane protein mitofilin, peroxiredoxin isoform PRX‐III, ATPase synthase, succinate dehydrogenase, mitochondrial fission protein Fis1, succinate‐coenzyme A ligase, acyl‐coenzyme A dehydrogenase, porin isoform VDAC2, ubiquinol‐cytochrome c reductase core I protein and prohibitin. Immunoblotting, enzyme testing and confocal microscopy were used to validate proteomic findings. The DIGE‐identified increase in key mitochondrial elements during aging agrees with the concept that sarcopenia is associated with a shift to a slower contractile phenotype and more pronounced aerobic‐oxidative metabolism. This suggests that mitochondrial markers are reliable candidates that should be included in the future establishment of a biomarker signature of skeletal muscle aging.     

Proteomic analysis of the hyaloid vascular system regression during ocular development

We describe a proteomic approach to investigate the differential protein expression patterns and identify the physiologically relevant angiogenic and antiangiogenic factors involved in the hyaloid vascular system regression. Differentially expressed proteins were identified using two-dimensional gel electrophoresis followed by nanoflow chromatography coupled with tandem mass spectrometry. These proteins are expected to provide insight as to their function in the early maintenance and eventual regression of the hyaloid vascular system.     

Proteomic analysis of human follicular fluid from fertile women

Background

Follicular fluid is a unique biological fluid in which the critical events of oocyte and follicular maturation and somatic cell-germ cell communication occur. Because of the intimate proximity of follicular fluid to the maturing oocyte, this fluid provides a unique window into the processes occurring during follicular maturation. A thorough identification of the specific components within follicular fluid may provide a better understanding of intrafollicular signaling, as well as reveal potential biomarkers of oocyte health for women undergoing assisted reproductive treatment. In this study, we used high and low pH HPLC peptide separations followed by mass spectrometry to perform a comprehensive proteomic analysis of human follicular fluid from healthy ovum donors. Next, using samples from a second set of patients, an isobaric mass tagging strategy for quantitative analysis was used to identify proteins with altered abundances after hCG treatment.

Results

A total of 742 follicular fluid proteins were identified in healthy ovum donors, including 413 that have not been previously reported. The proteins belong to diverse functional groups including insulin growth factor and insulin growth factor binding protein families, growth factor and related proteins, receptor signaling, defense/immunity, anti-apoptotic proteins, matrix metalloprotease related proteins, and complement activity. In a quantitative analysis, follicular fluid samples from age-matched women undergoing in vitro fertilization oocyte retrieval were compared and 17 follicular fluid proteins were found at significantly altered levels (p < 0.05) between pre-hCG and post-hCG samples. These proteins belong to a variety of functional processes, including protease inhibition, inflammation, and cell adhesion.

Conclusions

This database of FF proteins significantly extends the known protein components present during the peri-ovulatory period and provides a useful basis for future studies comparing follicular fluid proteomes in various fertility, disease, and environmental exposure conditions. We identified 17 differentially expressed proteins after hCG treatment and together these data showed the feasibility for defining biomarkers that illuminate how the ovarian follicle microenvironment is altered in various infertility-related conditions.

Electronic supplementary material

The online version of this article (doi:10.1186/s12014-015-9077-6) contains supplementary material, which is available to authorized users.     

Proteomic characterization of specific minor proteins in the human milk casein fraction

Human milk contains many bioactive proteins that are likely to support the early development of the newborn. The aim of this study was to identify whether there are specific minor proteins associated with the human milk casein micelle prepared by the acid precipitation method. Protein identification was performed by liquid chromatography tandem mass spectrometry analysis. Eighty-two proteins were identified in the casein micelle, 18 of which are not present in their whey compartment. Thirty-two of these proteins specifically associated with the casein micelle have not previously been identified in human milk or colostrum. Proteins involved in immune function comprised the major part (28%) of total proteins, and another significant part is involved in metabolism/energy production (22%). Most of the proteins were of extracellular or cytoplasmic origin (accounting for 50 and 29%, respectively). This study indicates that various soluble proteins should be considered as part of the casein compartment, prepared by the acid precipitation method. The data provide new insight not only into the proteomic profile of the human milk casein micelle and its physiological significance, but also into the proper proportion of casein and casein-associated proteins to use in infant formula.     

Proteomic tools to characterize the protein fraction of Equidae milk

The principal components of the protein fraction in pony mare's milk have been successfully identified and partially characterized using proteomic tools. Skimmed pony mare's milk was fractionated by either reversed phase-high-performance liquid chromatography (RP-HPLC) on a C4 column or a bi-dimensional separation technique coupling RP-HPLC in the first dimension and sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) in the second dimension (two-dimensional RP-HPLC/SDS-PAGE). The fractions thus obtained were analyzed by Edman N-terminal microsequencing and mass determination, with or without tryptic digestion, on a matrix-assisted laser desorption/ionization-time of flight spectrometer. Based on the sequence and molecular mass information obtained, identifications were achieved through a protein database search using homology or pattern research algorithms. This methodological approach was shown to be rapid, efficient and reliable in identifying the principal proteins in pony mare's milk. kappa-, alpha(s1)-, alpha(s2)-, and beta-casein, lysozyme C, alpha-lactalbumin and beta-lactoglobulin I and II were thus identified. alpha(s1) and beta-caseins displayed polymorphic patterns, probably due to alternative splicing processes leading to casual exon skipping events involving exons 7 and 14 in alpha(s1)-casein and exon 5 in beta-casein. Edman N-terminal microsequencing over 35 amino acid residues, for pony alpha(s1)-casein, clearly demonstrated the occurrence, in Equidae, of a splicing pattern similar to that reported in rodents, characterized by the constitutive outsplicing of exon 5. Pony mare's milk SDS-PAGE and RP-HPLC patterns were compared with those obtained for other milks (cow, goat and human), as were the relative levels of caseins and major whey proteins in these milks. Our results provide further evidence to support the notion that Equidae milk is closer to human breast milk than milk from bovine and caprine with respect to the casein and lysozyme C contents and casein/whey proteins ratio.     

Proteomic identification of collagens and related proteins in human fibroblasts

Summary. Fibroblasts are used for diagnosis of a series of metabolic diseases and are particularly suitable for the diagnosis of collagen disorders. We aimed to generate a skin fibroblast map that would be suitable for the concomitant determination of collagen and collagen-related proteins.A human skin fibroblast cell line was cultivated, homogenised, proteins extracted and subject to two-dimensional gel electrophoresis with subsequent in-gel-digestion of protein spots and mass spectrometrical identification (MALDI-TOF).Collagen alpha1 (I) chain precursor, collagen alpha1 (III) chain precursor, collagen alpha2 (VI) precursor and collagen modifying enzymes prolyl 4-hydroxylase alpha-2-subunit precursor, procollagen-lysine 2-oxoglutarate 5-dioxygenase 1 and 2, protein disulfide isomerase ER-60 precursor and peptidyl-prolyl cis-trans isomerase were among the abundant proteins.The finding of collagen and collagen-related structures as well as the identification of other metabolic enzyme systems on one 2D gel may propose the use of this proteomic method for further characterization of collagen and collagen-related proteins or for preliminary screening of metabolic disorders.     

Proteomic analysis of the response of human lung cells to uranium

The industrial use of uranium and particularly of depleted uranium, has pinpointed the need to review its chemical impact on human health. A proteomic approach was used to evaluate the response of a human lung cell line (A549) to uranium. We established the first 2-D reference map of the A549 cell line, identifying 87 spots corresponding to 81 major proteins. Uranium treatment triggered differential expression of 18 spots, of which 14 corresponded to fragments of cytokeratin 8 (CK8) and cytokeratin (CK18) and 1 to peroxiredoxin 1. We probed several hypotheses regarding CK cleavage, and observed that it did not result from caspase or calpain activity. Furthermore, we showed that the fragments are recognised by an anti-ubiquitin antibody (KM691). These results suggest a regulatory pathway involving CK ubiquitinylation or dysfunction in the proteasome-ubiquitin system in response to uranium exposure in human lung cells.     

Proteomic analysis of the resistance to aplidin in human cancer cells

Aplidin (plitidepsin) is an antitumoral agent that induces apoptosis via Rac1-JNK activation. A proteomic approach using 2D-DIGE technology found 52 cytosolic and 39 membrane proteins differentially expressed in wild-type and Aplidin-resistant HeLa cells, of which 39 and 27 were identified by MALDI-TOF mass spectrometry and database interrogation. A number of proteins involved in apoptosis pathways were found to be deregulated. Alterations in Rab geranylgeranyltransferase, protein disulfide isomerase (PDI), cystathionine gamma-lyase, ezrin, and cyclophilin A (CypA) were confirmed by immunoblotting. Moreover, the role of PDI and CypA in Aplidin resistance was functionally confirmed by using the inhibitor bacitracin and overexpression, respectively. These deregulated proteins are candidates to mediate, at least partially, Aplidin action and might provide a route to the cells to escape the induction of apoptosis by this drug.     

Proteomic analysis of lipid microdomains from lipopolysaccharide-activated human endothelial cells

The endothelium plays a critical role in orchestrating the inflammatory response seen during sepsis. Many of the inflammatory effects of Gram-negative sepsis are elicited by lipopolysaccharide (LPS), a glycolipid component of bacterial cell walls. Lipid-rich microdomains have been shown to concentrate components of the LPS signaling system. However, much remains to be learned about which proteins are constituents of lipid microdomains, and how these are regulated following cell activation. Progress in this area would be accelerated by employing global proteomic analyses, but the hydrophobicity of membrane proteins presents an analytical barrier to the effective application of such approaches. Herein, we describe a method to isolate detergent-resistant membranes from endothelial cells, and prepare these samples for proteomic analysis in a way that is compatible with subsequent separations and mass spectrometric (MS) analysis. In the application of these sample preparation and MS analyses, 358 proteins from the lipid-rich microdomains of LPS-activated endothelial cell membranes have been identified of which half are classified as membrane proteins by Gene Ontology. We also demonstrate that the sample preparation method used for solubilization and trypsin digestion of lipid-rich microdomains renders the membrane spanning sequences of transmembrane proteins accessible for endoproteolytic hydrolysis. This analysis sets the analytical foundation for an in-depth probing of LPS signaling in endothelial cells.     

Proteomic analysis of microvesicles derived from human mesenchymal stem cells

Mesenchymal stem cells (MSCs) have emerged as a promising means for treating degenerative or incurable diseases. Recent studies have shown that microvesicles (MVs) from MSCs (MSC-MVs) contribute to recovery of damaged tissues in animal disease models. Here, we profiled the MSC-MV proteome to investigate their therapeutic effects. LC-MS/MS analysis of MSC-MVs identified 730 MV proteins. The MSC-MV proteome included five positive and two variable known markers of MSCs, but no negative marker, as well as 43 surface receptors and signaling molecules controlling self-renewal and differentiation of MSCs. Functional enrichment analysis showed that cellular processes represented by the MSC-MV proteins include cell proliferation, adhesion, migration, and morphogenesis. Integration of MSC's self-renewal and differentiation-related genes and the proteome of MSC-conditioned media (MSC-CM) with the MSC-MV proteome revealed potential MV protein candidates that can be associated with the therapeutic effects of MSC-MVs: (1) surface receptors (PDGFRB, EGFR, and PLAUR); (2) signaling molecules (RRAS/NRAS, MAPK1, GNA13/GNG12, CDC42, and VAV2); (3) cell adhesion (FN1, EZR, IQGAP1, CD47, integrins, and LGALS1/LGALS3); and (4) MSC-associated antigens (CD9, CD63, CD81, CD109, CD151, CD248, and CD276). Therefore, the MSC-MV proteome provides a comprehensive basis for understanding the potential of MSC-MVs to affect tissue repair and regeneration.