Deeper in the human cornea proteome using nanoLC-Orbitrap MS/MS: An improvement for future studies on cornea homeostasis and pathophysiology

The cornea is a transparent, avascular, and highly specialized connective tissue that provides the majority of light refraction in the optical system of the eye. The human cornea is composed of several layers interacting in a complex manner and possessing specific functions, like eye protection and optical clearness. Only few proteomic studies of mammalian cornea have been performed leading to the identification of less than 200 proteins in human corneas. The present study explores the proteome of the intact normal human cornea using a shot-gun nanoLC-MS/MS strategy and an LTQ Orbitrap mass spectrometer. A total of 2070 distinct corneal proteins were identified from five human cornea samples, which represents a 14-fold improvement in the number of proteins identified so far for human cornea. This enlarged dataset of human corneal proteins represents a valuable reference library for further studies on cornea homeostasis and pathophysiology. Network and gene ontology analyses were used to determine biological pathways specific of the human cornea. They allowed the identification of subnetworks of putative importance for corneal diseases, like a redox regulation and oxidative stress network constituted of aldehyde and alcohol dehydrogenases, most of them being described for the first time in human cornea.     

Human cornea proteome: identification and quantitation of the proteins of the three main layers including epithelium, stroma, and endothelium

Diseases of the cornea are common and refer to conditions like infections, injuries and genetic defects. Morphologically, many corneal diseases affect only certain layers of the cornea and separate analysis of the individual layers is therefore of interest to explore the basic molecular mechanisms involved in corneal health and disease. In this study, the three main layers including, the epithelium, stroma and endothelium of healthy human corneas were isolated. Prior to analysis by LC-MS/MS the proteins from the different layers were either (i) separated by SDS-PAGE followed by in-gel trypsinization, (ii) in-solution digested without prior protein separation or, (iii) in-solution digested followed by cation exchange chromatography. A total of 3250 unique Swiss-Prot annotated proteins were identified in human corneas, 2737 in the epithelium, 1679 in the stroma, and 880 in the endothelial layer. Of these, 1787 proteins have not previously been identified in the human cornea by mass spectrometry. In total, 771 proteins were quantified, 157 based on in-solution digestion and 770 based on SDS-PAGE separation followed by in-gel digestion of excised gel pieces. Protein analysis showed that many of the identified proteins are plasma proteins involved in defense responses.     

Expanding the proteome two-dimensional gel electrophoresis reference map of human renal cortex by peptide mass fingerprinting

Proteomics methodologies hold great promise in basic renal research and clinical nephrology. The classical approach for proteomic analysis couples two-dimensional gel electrophoresis (2-DE) with protein identification by mass spectrometry, to produce more global information regarding normal protein expression and alterations in different physiological and pathological states. In this report we have expanded the identification of proteins in the renal cortex, improving the previously published map to facilitate the study of different diseases affecting the human kidney. About 250 spots were analyzed by peptide mass fingerprinting, 89 proteins and 74 isoforms for some of them were identified and implemented in the normal human renal cortex 2-DE reference map. This more comprehensive view of the proteome of the human renal cortex could be of invaluable help to the differential proteomic display of urological diseases.     

Changes of extracellular matrix of rat cornea after exposure to hypoxia

The purpose of the study was to check whether hypoxia of corneal tissue increases the collagenolytic activity due to release of reactive oxygen and nitrogen species. Rats were exposed to hypoxia 10% O(2) for 4, 14, and 21 days. The radical tissue injury was measured by the level of nitrotyrosine and changes in the lipoperoxide-related fluorophores. Collagen protein composition was analyzed by slab gel electrophoresis. The activity of gelatinolytic enzymes was studied using the zymography. The vascularization of the corneas was measured. We found no differences in the corneal tissue in the gel electrophoretic profile of collagenous proteins and gelatinolytic activity between normoxic and hypoxic rats. We did not find any sign of radical tissue injury. There were no changes in the vascularization of corneas after exposition to hypoxia. The environmental 10% hypoxia does not induce radical tissue injury and an increase of collagenolytic activity in the rat cornea.     

In-depth analysis of the human CSF proteome using protein prefractionation

The identification of disease markers in human body fluids requires an extensive and thorough analysis of its protein constituents. In the present study, we have extended our analysis of the human cerebrospinal fluid (CSF) proteome using protein prefractional followed by shotgun mass spectrometry. After the removal of abundant protein components from the mixture with the help of immunodepletion affinity chromatography, we used either anion exchange chromatography or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to further subfractionate the proteins present in CSFs. Each protein subfraction was enzyme digested and analyzed by tandem mass spectrometry and the resulting data evaluated using the Spectrum Mill software. Different subfractionation methods resulted in the identification of a grant total of 259 proteins in CSF from a patient with normal pressure hydrocephalus. The greatest number of protein, 240 in total, were identified after prefractionating the CSF proteins by immunodepletion and SDS-PAGE. Immuno-depletion combined with anion exchange fractionation resulted in 112 proteins and 74 proteins were found when only immunodepletion of the CSF samples was carried out. All methods used showed a significant increase in the number of identified proteins as compared with nondepleted and unfractionated CSF sample analysis, which yielded only 38 protein identifications. The present work establishes a platform for future studies aimed at a detailed comparative proteome analysis of CSFs from different groups of patients suffering from various psychiatric and neurological disorders.     

The role of mass spectrometry in proteome studies

Mass spectrometry (MS) is an important tool in modern protein chemistry. In proteome analyses the expression of hundreds or thousands of proteins can be monitored at the same time. First, complex protein mixtures are separated by two-dimensional gel electrophoresis (2-DE) and then individual proteins are identified by using MS followed by database searches. Recent developments in this field have made it possible to do automated, high-throughput protein identification that is needed in proteome analyses. MS can also be used to characterize post-translational modifications in proteins and to study protein complexes. This review will introduce the current MS methods used in proteome studies, and discuss their advantages and disadvantages. New instrumental MS developments are also presented that are useful in these analyses.     

Comparative proteome analysis of breast cancer and normal breast

Breast cancer is a leading cause of death for women. The underlying molecular mechanism is still not well understood. In this study, two-dimensional gel electrophoresis combined with mass spectrometry was used to analyze changes in the proteome of infiltrating ductal carcinoma compared to normal breast tissue. Ten sets of two-dimensional gels per experimental condition were analyzed and more than 500 spots each were detected. This revealed 39 spots for which expression in breast cancer cells were reproducibly altered more than twofold compared to normal controls (p<0.01). These spots represented 25 different proteins after identification using the database search after mass spectrometry, comprising cell defense proteins, enzymes involved in glycolytic energy metabolism and homeostasis, protein folding and structural proteins, proteins involved in cytoskeleton and cell motility, and proteins involved in other functions. In addition, 28 nondifferentially expressed proteins with different functions were also mapped and identified, which might help to establish a two-dimensional gel electrophoresis reference map of human breast cancer. Our study shows that proteomics offers a powerful methodology to detect the proteins that show different expression patterns in breast cancer tissue and may provide an accurate molecular classification. The differentially expressed proteins may be used as potential candidate markers for diagnostic purposes or for determination of tumor sensitivity to therapy. The functional implications of the identified proteins are discussed.     

High resolution mass spectrometric alveolar proteomics: identification of surfactant protein SP-A and SP-D modifications in proteinosis and cystic fibrosis patients

In the present study, one- and two-dimensional gel electrophoresis combined with high resolution Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) have been applied as powerful approaches for the proteome analysis of surfactant proteins SP-A and SP-D, including identification of structurally modified and truncation forms, in bronchoalveolar lavage fluid from patients with cystic fibrosis, chronic bronchitis and pulmonary alveolar proteinosis. Highly sensitive micropreparation techniques were developed for matrix-assisted laser desorption/ionization (MALDI) FT-ICR MS analysis which provided the identification of surfactant proteins at very low levels. Owing to the high resolution, FT-ICR MS was found to provide substantial advantages for the structural identification of surfactant proteins from complex biological matrices with high mass determination accuracy. Several protein bands corresponding to SP-A and SP-D were identified by MALDI-FT-ICR MS after electrophoretic separation by one- and two-dimensional gel electrophoresis, and provided the identification of structural modifications (hydroxy-proline) and degradation products. The high resolution mass spectrometric proteome analysis should facilitate the unequivocal identification of subunits, aggregations, modifications and degradation products of surfactant proteins and hence contribute to the understanding of the mechanistic basis of lung disease pathogenesis.     

Establishment of a root proteome reference map for the model legume Medicago truncatula using the expressed sequence tag database for peptide mass fingerprinting

We have established a proteome reference map for Medicago truncatula root proteins using two-dimensional gel electrophoresis combined with peptide mass fingerprinting to aid the dissection of nodulation and root developmental pathways by proteome analysis. M. truncatula has been chosen as a model legume for the study of nodulation-related genes and proteins. Over 2,500 root proteins could be displayed reproducibly across an isoelectric focussing range of 4-7. We analysed 485 proteins by peptide mass fingerprinting, and 179 of those were identified by matching against the current M. truncatula expressed sequence tag (EST) database containing DNA sequences of approximately 105,000 ESTs. Matching the EST sequences to available plant DNA sequences by BLAST searches enabled us to predict protein function. The use of the EST database for peptide identification is discussed. The majority of identified proteins were metabolic enzymes and stress response proteins, and 44% of proteins occurred as isoforms, a result that could not have been predicted from sequencing data alone. We identified two nodulins in uninoculated root tissue, supporting evidence for a role of nodulins in normal plant development. This proteome map will be updated continuously (http://semele.anu.edu.au/2d/2d.html) and will be a powerful tool for investigating the molecular mechanisms of root symbioses in legumes.     

Proteomics of the aqueous humor in healthy New Zealand rabbits

There are several physiological roles postulated for aqueous humor, a liquid located in the anterior and posterior chamber of the eye, such as maintenance of the intraocular pressure, provision of nutrients, and removal of metabolic waste from neighboring tissues and provision of an immune response and protection during inflammation and infection. To link these function to specific or classes of proteins, identification of the aqueous humor proteome is essential. Aqueous humor obtained from healthy New Zealand white rabbits was analyzed using three synergistic protein separation methods: 1-D gel electrophoresis, 2-DE, and 1-DLC (RPLC) prior to protein identification by MS. As each of these separation methods separates intact proteins based on different physical properties (pIs, molecular weights, hydrophobicity, solubility, etc.) the proteome coverage is expanded. This was confirmed, since overlap between all three separation technologies was only about 8.2% with many proteins found uniquely by a single method. Although the most dominant protein presented in normal aqueous humor is albumin, by using this extensive separation/MS strategy, additional proteins were identified in total amount of 98 nonredundant proteins (plus an additional ten proteins for consideration). This expands the current protein identifications by approximately 65%. The aqueous humor proteome comprises a specific selection of cellular and plasma based proteins and can almost exclusively be divided into four functional groups: cell-cell interactions/wound healing, proteases and protease inhibitors, antioxidant protection, and antibacterial/anti-inflammatory proteins.     

Extensive analysis of the human platelet proteome by two-dimensional gel electrophoresis and mass spectrometry

Platelets play a key role in the control of bleeding and wound healing, contributing to the formation of vascular plugs. Under pathologic circumstances, they are involved in thrombotic disorders, including heart disease. Since platelets do not have a nucleus, proteomics offers a powerful alternative approach to provide data on protein expression in these cells, helping to address their biology. In this publication we extend the previously reported analysis of the pI 4-5 region of the human platelet proteome to the pI 5-11 region. By using narrow pI range two-dimensional electrophoresis (2-DE) for protein separation followed by high-throughput tandem mass spectrometry (MS/MS) for protein identification, we were able to identify 760 protein features, corresponding to 311 different genes, resulting in the annotation of 54% of the pI 5-11 range 2-DE proteome map. We evaluated the physicochemical properties and functions of the identified platelet proteome. Importantly, the main group of proteins identified is involved in intracellular signalling and regulation of the cytoskeleton. In addition, 11 hypothetical proteins are reported. In conclusion, this study provides a unique inventory of the platelet proteome, contributing to our understanding of platelet function and building the basis for the identification of new drug targets.     

Proteoglycan biosynthesis by human corneas from patients with types 1 and 2 macular corneal dystrophy

Corneal buttons were obtained from patients with types 1 and 2 macular corneal dystrophy (MCD) and from control patients with Fuchs' dystrophy or keratoconus. Buttons were incubated for 20 h in the presence of [3H]glucosamine or [2-3H]mannose. Radiolabeled proteoglycans and lactosaminoglycan-glycoproteins (L-GPs) were purified using chromatography on Q-Sepharose, Superose 6, and octyl-Sepharose. They were identified using chondroitinase ABC, keratanase or endo-beta-galactosidase digestion, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis or Superose 6 chromatography. This study confirms previous reports that type 1 MCD corneas synthesize a normal dermatan sulfate-proteoglycan (DS-PG) and an abnormal keratan sulfate-proteoglycan (KS-PG). The data indicate that typ 1 MCD corneas synthesize L-GP instead of KS-PG. This L-GP has a core protein of similar hydrophobicity (elution from octyl-Sepharose) and nearly similar mass (42 kDa) as the core protein of the KS-PG. It has identical glycoconjugates as those of the KS-PG except that they lack sulfate. Thus, type 1 MCD fails to synthesize keratan sulfate as a result of a defect in a sulfotransferase specific for sulfating lactosaminoglycans. Further, proteoglycans synthesized by a cornea from a patient with type 2 MCD were studied. This cornea synthesized a normal ratio of KS-PG to DS-PG although net synthesis of proteoglycans was approximately 30% below normal. The KS-PG appeared normal whereas the DS-PG had dermatan sulfate chains that were approximately 40% shorter than normal.     

Selection of the peptide mass tolerance value for protein identification with peptide mass fingerprinting

Peptide mass fingerprinting (PMF) is widely used for protein identification while studying proteome via time-of-flight mass spectrometer or via 1D or 2D electrophoresis. Peptide mass tolerance indicating the fit of theoretical peptide mass to an experimental one signifcantly influences protein identification. The role of peptide mass tolerance could be estimated by counting the number of correctly identified proteins for the reference set of mass spectra. The reference set of 400 Ultraflex (Bruker Daltonics, Germany) protein mass spectra was obtained for liver microsomes slices hydrolyzed via 1D gel electrophoresis. Using a Mascot server for protein identification, the peptide mass tolerance value varied within 0.02–0.40 Da with a step of 0.01 Da. The number of identified proteins changed up to 10 times depending on the tolerance. The maximal number of identified proteins was reported for the tolerance value of 0.15 Da (120 ppm) known to be 1.5–2-fold higher than the recommended values for such a type of mass spectrometer. The software program PMFScan was developed to obtain the dependence between the number of identified proteins and the tolerance values.     

Proteome profiling of human epithelial ovarian cancer cell line TOV-112D

A proteome profiling of the epithelial ovarian cancer cell line TOV-112D was initiated as a protein expression reference in the study of ovarian cancer. Two complementary proteomic approaches were used in order to maximise protein identification: two-dimensional gel electrophoresis (2DE) protein separation coupled to matrix assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and one-dimensional gel electrophoresis (1DE) coupled to liquid-chromatography tandem mass spectrometry (LC MS/MS). One hundred and seventy-two proteins have been identified among 288 spots selected on two-dimensional gels and a total of 579 proteins were identified with the 1DE LC MS/MS approach. This proteome profiling covers a wide range of protein expression and identifies several proteins known for their oncogenic properties. Bioinformatics tools were used to mine databases in order to determine whether the identified proteins have previously been implicated in pathways associated with carcinogenesis or cell proliferation. Indeed, several of the proteins have been reported to be specific ovarian cancer markers while others are common to many tumorigenic tissues or proliferating cells. The diversity of proteins found and their association with known oncogenic pathways validate this proteomic approach. The proteome 2D map of the TOV-112D cell line will provide a valuable resource in studies on differential protein expression of human ovarian carcinomas while the 1DE LC MS/MS approach gives a picture of the actual protein profile of the TOV-112D cell line. This work represents one of the most complete ovarian protein expression analysis reports to date and the first comparative study of gene expression profiling and proteomic patterns in ovarian cancer.     

Selection of the peptide mass tolerance value for the protein identification with peptide mass fingerprinting

Peptide mass-fingerprint is widely used for protein identification while studying proteome with the use of 1D or 2D electrophoresis. Peptide mass tolerance indicates the fit of theoretical peptide mass with the experimental measurements, and choice of this parameter sufficiently influences the protein identification. The role of peptide mass tolerance was estimated by counting the number of identified proteins for the reference set of mass-spectra. The reference set of 400 Ultraflex (Bruker Daltonics, Germany) mass-spectra was obtained for the slices of 1D gel of liver microsomes. Using Mascot server for protein identification, the peptide mass tolerance value was varied in the range from 0.02 to 0.40 Da with a step 0.01 Da. Depending on the tolerance the number of identified protein changes up to 10 times. Maximal number of identified proteins was reported for the tolerance value of 0.15 Da (120 ppm), which is 1.5 - 2 times higher than the recommended values for such type of mass-spectrometers. The software program PMFScan was developed to obtain the dependence of number of identified proteins of the tolerance values.     

Human plasma proteome analysis by reversed sequence database search and molecular weight correlation based on a bacterial proteome analysis

In shotgun proteomics, proteins can be fractionated by 1-D gel electrophoresis and digested into peptides, followed by liquid chromatography to separate the peptide mixture. Mass spectrometry generates hundreds of thousands of tandem mass spectra from these fractions, and proteins are identified by database searching. However, the search scores are usually not sufficient to distinguish the correct peptides. In this study, we propose a confident protein identification method for high-throughput analysis of human proteome. To build a filtering protocol in database search, we chose Pseudomonas putida KT2440 as a reference because this bacterial proteome contains fewer modifications and is simpler than the human proteome. First, the P. putida KT2440 proteome was filtered by reversed sequence database search and correlated by the molecular weight in 1-D-gel band positions. The characterization protocol was then applied to determine the criteria for clustering of the human plasma proteome into three different groups. This protein filtering method, based on bacterial proteome data analysis, represents a rapid way to generate higher confidence protein list of the human proteome, which includes some of heavily modified and cleaved proteins.     

Review of application of mass spectrometry for analyses of anterior eye proteome

Proteins have important functional roles in the body, which can be altered in disease states. The eye is a complex organ rich in proteins; in particular, the anterior eye is very sophisticated in function and is most commonly involved in ophthalmic diseases. Proteomics, the large scale study of proteins, has greatly impacted our knowledge and understanding of gene function in the post-genomic period. The most significant breakthrough in proteomics has been mass spectrometric identification of proteins, which extends analysis far beyond the mere display of proteins that classical techniques provide. Mass spectrometry functions as a "mass analyzer" which simplifies the identification and quantification of proteins extracted from biological tissue. Mass spectrometric analysis of the anterior eye proteome provides a differential display for protein comparison of normal and diseased tissue. In this article wepresent the key proteomic findings in the recent literature related to the cornea, aqueous humor, trabecular meshwork, iris, ciliary body and lens. Through this we identified unique proteins specific to diseases related to the anterior eye.