Identification of a new potential airway irritation marker, palate lung nasal epithelial clone protein, in human nasal lavage fluid with two-dimensional electrophoresis and matrix-assisted laser desorption/ionization-time of flight.

We have analyzed protein patterns of human nasal lavage fluid (NLF) with two-dimensional gel electrophoresis (2-DE) and identified several proteins (such as transthyretin, Clara Cell protein 16, lipocalin-1, cystatin S, cystatin SN, immunoglobulin binding factor, statherin, calgranulin B, prolactin-inducible protein, and zinc-alpha2-glycoprotein) by N-terminal amino acid sequencing and matrix-assisted laser desorption/ionizationtime of flight (MALDI-TOF) mass spectrometry. To investigate whether airway irritation causes alterations in NLF 2-DE patterns, we compared epoxy workers with airway irritation (n=8) and healthy controls (n=6) before and after 2 h exposure to the epoxy chemical, dimethylbenzylamine (DMBA, 100 microg/m3) in an exposure chamber. A 25 kDa protein with pI 5.5 was found to be altered in the NLF 2-DE patterns; a trypsin digest of the 2-DE spot analyzed by MALDI-TOF and expressed sequence tags (ESTs) determined after post-source decay (PSD) identified the protein as palate lung and nasal epithelial clone (PLUNC). In controls, the levels of NLF-PLUNC were generally lower after 2 h exposure, whereas in epoxy workers, the levels were increased three- to twentyfold after exposure. The human gene sequence for PLUNC was just recently reported and so far no biofunctional data are available. Our results suggest that PLUNC is involved in the airway inflammatory response after exposure to irritants.     

Sequence‐Defined Heteromultivalent Precision Glycomacromolecules Bearing Sulfonated/Sulfated Nonglycosidic Moieties Preferentially Bind Galectin‐3 and Delay Wound Healing of a Galectin‐3 Positive Tumor Cell Line in an In Vitro Wound Scratch Assay

Within this work, a new class of sequence‐defined heteromultivalent glycomacromolecules bearing lactose residues and nonglycosidic motifs for probing glycoconjugate recognition in carbohydrate recognition domain (CRD) of galectin‐3 is presented. Galectins, a family of β‐galactoside‐binding proteins, are known to play crucial roles in different signaling pathways involved in tumor biology. Thus, research has focused on the design and synthesis of galectin‐targeting ligands for use as diagnostic markers or potential therapeutics. Heteromultivalent precision glycomacromolecules have the potential to serve as ligands for galectins. In this work, multivalency and the introduction of nonglycosidic motifs bearing either neutral, amine, or sulfonated/sulfated groups are used to better understand binding in the galectin‐3 CRD. Enzyme‐linked immunosorbent assays and surface plasmon resonance studies are performed, revealing a positive impact of the sulfonated/sulfated nonglycosidic motifs on galectin‐3 binding but not on galectin‐1 binding. Selected compounds are then tested with galectin‐3 positive MCF 7 breast cancer cells using an in vitro would scratch assay. Preliminary results demonstrate a differential biological effect on MCF 7 cells with high galectin‐3 expression in comparison to an HEK 293 control with low galectin‐3 expression, indicating the potential for sulfonated/sulfated heteromultivalent glycomacromolecules to serve as preferential ligands for galectin‐3 targeting.     

Quantitative assessment of human serum high-abundance protein depletion

The aim of this study is to quantify the effectivity of the depletion of human high-abundance serum and plasma proteins for improved protein identification and disease marker candidate discovery and to assess the risk of concomitant removal of relevant marker proteins. 2-DE and bottom-up shotgun MS combining 2-D capillary chromatography with MS/MS were applied in parallel for the analysis of fractions resulting from the depletion procedure. For many proteins the factors of enrichment by the depletion were obvious allowing their enhanced detection and identification upon high-abundance protein depletion. Nano-liquid chromatography linked MS allowed the efficient identification of several low-abundant proteins that were not identified on the 2-DE gels. Resolving the fractions that were eluted from the matrix upon depletion indicated unspecific binding of disease relevant proteins in plasma samples from acute myocardial infarction patients. The unspecific binding to the depletion matrix of inflammatory markers spiked into the serum was found to depend on the type of capturing agent used. Polyclonal avian antibodies (IgY) displayed the least unspecific binding due to the high immunogenicity of mammalian proteins in avian hosts.     

Mass spectrometry meets chip technology: a new proteomic tool in cancer research?

DNA chip technologies are the most exiting genomic tools, which were developed within the last few years. It is, however, evident that knowledge of the gene sequence or the quantity of gene expression is not sufficient to predict the biological nature and function of a protein. This can be particularly important in cancer research where post-translational modifications of a protein can specifically contribute to the disease. To address this problem, several proteomic tools have been developed. Currently the most widely used proteomic tool is two-dimensional protein gel electrophoresis (2-DE), which can display protein expression patterns to a high degree of resolution. As an alternative to 2-DE, a preliminary study using a new technique was employed to generate protein expression patterns from whole tissue extracts. Surface-enhanced laser desorption/ionization (SELDI) allows the retention of proteins on a solid-phase chromatographic surface (ProteinChip Array) with direct detection of retained proteins by time of flight-mass spectrometry (TOF-MS). Using this system, we analyzed eight cases of renal cell carcinoma (RCC) including normal, peripheral and central tumor tissue as well as four microdissected cases of cervical intraepithelial neoplasia (CIN) and three microdissected cases of cervix uteri carcinoma. Differentially expressed proteins were found by comparing the protein expression patterns generated using SELDI-based TOF-MS of tumor tissue with normal and neoplastic tissue, respectively. By applying this fast and powerful ProteinChip array technology it becomes possible to investigate complex changes at the protein level in cancer associated with tumor development and progression.     

Down-regulation of Vinculin in Human Colorectal Carcinoma Identified by Proteomics Analysis

In the present study, we aimed to globally profile the proteins involved in colorectal carcinoma(CRC), in order to find clues to the pathological process of CRC. Pairs of malignant tissues and their adjacent healthy tissues from patients with colorectal cancer were subject to differential proteomics analysis. Two dimensional electrophoresis coupled with mass spectrometry(2-DE/MS) was used to identify differentially expressed proteins between pairs of tissue samples. A list of proteins relevant to the progression of colorectal tumor was identified by two dimensional gel electrophoresis(2-DE)-based proteomics approach. Among the identified proteins, vinculin was found to be remarkably down-regulated in colorectal carcinoma tissues. In addition, three phosphorylation modifications within the isolated vinculin were identified by in-depth liquid chromatography-tandem mass spectrometry(LC-MS/MS) analysis. Our results provide a basis for further understanding the pathological significance of vinculin in the regulation of carcinogenesis, invasion and metastasis of colorectal tumors.     

A simple protocol for protein extraction of recalcitrant fruit tissues suitable for 2-DE and MS analysis

Fruit tissues are considered recalcitrant plant tissue for proteomic analysis. Three phenol-free protein extraction procedures for 2-DE were compared and evaluated on apple fruit proteins. Incorporation of hot SDS buffer, extraction with TCA/acetone precipitation was found to be the most effective protocol. The results from SDS-PAGE and 2-DE analysis showed high quality proteins. More than 500 apple polypeptides were separated on a small scale 2-DE gel. The successful protocol was further tested on banana fruit, in which 504 and 386 proteins were detected in peel and flesh tissues, respectively. To demonstrate the quality of the extracted proteins, several protein spots from apple and banana peels were cut from 2-DE gels, analyzed by MS and have been tentatively identified. The protocol described in this study is a simple procedure which could be routinely used in proteomic studies of many types of recalcitrant fruit tissues.     

Comprehensive proteome analysis of mouse liver by ampholyte-free liquid-phase isoelectric focusing

In this study, ampholyte-free liquid-phase IEF (LIEF) was combined with narrow pH range 2-DE and SDS-PAGE RP-HPLC for comprehensive analysis of mouse liver proteome. Because LIEF prefractionation was able to reduce the complexity of the sample and enhance the loading capacity of IEF strips, the number of visible protein spots on subsequent 2-DE gels was significantly increased. A total of 6271 protein spots were detected after integrating five narrow pH range 2-DE gels following LIEF prefractionation into a single virtual 2-DE gel. Furthermore, the pH 3-5 LIEF fraction and the unfractionated sample were separated by pH 3-6 2-DE and identified by MALDI-TOF/TOF MS, respectively. In parallel, the pH 3-5 LIEF fraction was also analyzed by SDS-PAGE RP-HPLC MS/MS. LIEF-2-DE and LIEF-HPLC could obviously improve the separation efficiency and the confidence of protein identification, which identified a higher number of low-abundance proteins and proteins with extreme physicochemical characteristics or post-translational modifications compared to conventional 2-DE method. Furthermore, there were 207 proteins newly identified in mouse liver in comparison with previously reported large-scale datasets. It was observed that the combination of LIEF-2-DE and LIEF-HPLC was effective in promoting MS-based liver proteome profiling and could be applied on similar complex tissue samples.     

Acid-labile surfactant improves in-sodium dodecyl sulfate polyacrylamide gel protein digestion for matrix-assisted laser desorption/ionization mass spectrometric peptide mapping

Mass spectrometry (MS) together with genome database searches serves as a powerful tool for the identification of proteins. In proteome analysis, mixtures of cellular proteins are usually separated by sodium dodecyl sulfate (SDS) polyacrylamide gel-based two-dimensional gel electrophoresis (2-DE) or one-dimensional gel electrophoresis (1-DE), and in-gel digested by a specific protease. In-gel protein digestion is one of the critical steps for sensitive protein identification by these procedures. Efficient protein digestion is required for obtaining peptide peaks necessary for protein identification by MS. This paper reports a remarkable improvement of protein digestion in SDS polyacrylamide gels using an acid-labile surfactant, sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate (ALS). Pretreatment of gel pieces containing protein spots separated by 2-DE with a small amount of ALS prior to trypsin digestion led to increases in the digested peptides eluted from the gels. Consistently, treatment of gel pieces containing silver-stained standard proteins and those separated from tissue extracts resulted in the detection of increased numbers of peptide peaks in spectra obtained by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOFMS). Hence the present protocol with ALS provides a useful strategy for sensitive protein identification by MS.     

Proteome analysis of human stomach tissue: separation of soluble proteins by two-dimensional polyacrylamide gel electrophoresis and identification by mass spectrometry

Two-dimensional gel electrophoresis (2-DE) maps for human stomach tissue proteins have been prepared by displaying the protein components of the tissue by 2-DE and identifying them using mass spectrometry. This will enable us to present an overview of the proteins expressed in human stomach tissues and lays the basis for subsequent comparative proteome analysis studies with gastric diseases such as gastric cancer. In this study, 2-DE maps of soluble fraction proteins were prepared on two gel images with partially overlapping pH ranges of 4-7 and 6-9. On the gels covering pH 4-7 and pH 6-9, about 900 and 600 protein spots were detected by silver staining, respectively. For protein identification, proteins spots on micropreparative gels stained with colloidal Coomassie Brilliant Blue G-250 were excised, digested in-gel with trypsin, and analyzed by peptide mass fingerprinting with delayed extraction-matrix assisted laser desorption/ionization-mass spectrometry (DE-MALDI-MS). In all, 243 protein spots (168 spots in acidic map and 75 spots in basic map) corresponding to 136 different proteins were identified. Besides these principal maps, overview maps (displayed on pH 3-10 gels) for total homogenate and soluble fraction, are also presented with some identifications mapped on them. Based on the 2-DE maps presented in this study, a 2-DE database for human stomach tissue proteome has been constructed and is available at http://proteome.gsnu.ac.kr/DB/2DPAGE/Stomach/. The 2-DE maps and the database resulting from this study will serve important resources for subsequent proteomic studies for analyzing the normal protein variability in healthy tissues and specific protein variations in diseased tissues.     

Enrichment of low-abundant serum proteins by albumin/immunoglobulin G immunoaffinity depletion under partly denaturing conditions

We present a simple protocol for affinity depletion to remove the two most abundant serum proteins, albumin and immunoglobulin G (IgG). Under native conditions, albumin/IgG were efficiently removed and several proteins were enriched as shown by two-dimensional electrophoresis (2-DE). Besides that, partly denaturing conditions were established by adding 5 or 20% acetonitrile (ACN) in order to disrupt the binding of low-molecular-weight (LMW) proteins to the carrier proteins albumin/IgG. 2-DE results showed that the total number of detected LMW proteins increased under denaturing conditions when compared to native conditions. Interestingly, the presence of 5% ACN in serum revealed better enrichment of LMW proteins when compared to 20% ACN condition. Seven randomly distributed spots in albumin/IgG depleted serum samples under 5% ACN condition were picked from the 2-DE gels and identified by mass spectrometry (MS). The intensity of five LMW protein spots increased under denaturing conditions when compared to native conditions. Three of the seven identified spots (serum amyloid P, vitamin D-binding protein, and transthyretin) belong to a group of relatively low-abundant proteins, which make up only 1% of all serum proteins. The method presented here improves the resolution of the serum proteome by increasing the number of visualized spots on 2-D gels and allowing the detection and MS identification of LMW proteins and proteins of lower abundance.     

Mass Spectrometric Characterization of Protein Structure Details Refines the Proteome Signature for Invasive Ductal Breast Carcinoma

Early diagnosis as well as individualized therapies are necessary to reduce the mortality of breast cancer, and personalized patient care strategies rely on novel prognostic or predictive factors. In this study, with six breast cancer patients, 2D gel analysis was applied for studying protein expression differences in order to distinguish invasive ductal breast carcinoma, the most frequent breast tumor subtype, from control samples. In total, 1203 protein spots were assembled in a 2D reference gel. Differentially abundant spots were subjected to peptide mass fingerprinting for protein identification. Twenty proteins with their corresponding 38 differentially expressed 2D gel spots were contained in our previously reported proteome signature, suggesting that distinct protein forms were contributing. In-depth MS/MS measurements enabled analyses of protein structure details of selected proteins. In protein spots that significantly contributed to our signature, we found that glyceraldehyde-3-phosphate dehydrogenase was N-terminally truncated, pyruvate kinase M2 and nucleoside diphosphate kinase A but not other isoforms of these proteins were of importance, and nucleophosmin phosphorylation at serine residues 106 and 125 were clearly identified. Principle component analysis and hierarchical clustering with normalized quantitative data from the 38 spots resulted in accurate separation of tumor from control samples. Thus, separation of tissue samples as in our initial proteome signature could be confirmed even with a different proteome analysis platform. In addition, detailed protein structure investigations enabled refining our proteome signature for invasive ductal breast carcinoma, opening the way to structure-/function studies with respect to disease processes and/or therapeutic intervention.     

Biomedical applications of two-dimensional electrophoresis using immobilized pH gradients: current status

There is currently much interest, as we enter the postgenome era, in studying gene expression at the protein level. Two-dimensional electrophoresis (2-DE) using immobilized pH gradients (IPG), coupled with mass spectrometry (MS), is currently the most widely utilized approach for the analysis of whole tissue proteins. The methodology for IPG-based 2-DE, since the introduction of the technique in the 1980s, is reviewed. In its present form the IPG methodology is mostly useful as a research tool. In general, high reproducibility and high resolution have been achieved. However, the lack of substantial automation and the limited sensitivity of the current overall methodology continue to represent drawbacks for biomedical applications. Further developments to increase throughput and to reduce sample requirement would substantially benefit the application of IPG-based 2-DE to biomedicine and would enhance the prospects for introducing the methodology into the clinical laboratory.     

Two-dimensional polyacrylamide gel electrophoresis of human lung cancer: qualitative aspects of tissue preparation in relation to histopathology

It is of great importance to identify new objective markers significant for the diagnosis, treatment, and prognosis of human cancers. Cytometric assessment of the DNA content has been shown to be one parameter which correlates excellently with the clinical course of patients with solid cancers of the breast, prostate, and thyroid. However, additional specific marker polypeptides are needed in order to further improve the diagnostic and prognostic sensitivity. Two-dimensional polyacrylamide gel electrophoresis (2-DE) is a unique tool in this field of cancer research, but serious problems concerning intersample variations in the 2-DE pattern must be solved because quantitative variations of potential marker polypeptides are expected to occur at low levels. In this study we examined a modified preparation method for tumor tissues, focusing on the relation between histopathological properties and 2-DE gel quality. We selected a group of human lung cancers sharing similar prognosis; nevertheless, the 2-DE patterns showed significant intersample but low intrasample variations. It is concluded that histopathological features, such as a local homogeneity, and the amounts of connective tissue and serum proteins are critical factors for the successful preparation and high quality of 2-DE-gels.     

Acute‐phase proteins investigation based on lectins affinity capture prior to 2‐DE separation: Application to serum from multiple sclerosis patients

Plasma acute‐phase proteins (APPs) glyco‐isoforms are important biomarkers of inflammatory processes such as those occurring in multiple sclerosis (MS). Specific analysis of these proteins is often hampered by sample biochemical complexity. The aim of our study was to set up a method to accurately visualize, identify and quantify APPs glyco‐isoforms in human serum. An enrichment strategy based on affinity chromatography using the carbohydrate‐binding proteins concanavalin A (ConA) and erythrina cristagalli lectin (ECL) was applied to pooled serum samples from 15 patients and 9 healthy individuals. Image analysis of 2‐DE detected 30 spots with a fold change higher than 1.5. A total of 14 were statistically significant (p value<0.05): 7 up‐regulated and 7 down‐regulated in MS samples. ESI LC‐Nanospray IT mass spectrometry analysis confirmed that all of them were APPs isoforms supporting the idea that the accurate analysis of differential glycosylation profiles in these biomarkers is instrumental to distinguish between MS patients and healthy subjects. Additionally, overlaps in ConA/ECL maps protein patterns suggest how the used lectins are able to bind sugars harbored by the same oligosaccharide structure. Among identified proteins, the presence of complex and/or hybrid type N‐linked sugar structures is well known. Performing galectin‐3 binding and Western blotting, we were able to demonstrate a correlation between hybrid type glyco‐isoforms of β‐haptoglobin and MS. In conclusion, although the patho‐physiological role of the identified species still remains unclear and further validations are needed, these findings may have a relevant impact on disease‐specific marker identification approaches.     

Thermal denaturation produced degenerative proteins and interfered with MS for proteins dissolved in lysis buffer in proteomic analysis

In 1-DE, proteins were traditionally mixed with the standard Laemmli buffer and boiled for several minutes. Recently, proteins dissolved in lysis buffer were used to produce better-resolved 2-DE gels, but thermal denaturation procedure still remained in some proteomic analysis. To determine the detailed effects of thermal denaturation on SDS-PAGE and MS, both 1-DE and 2-DE were performed using proteins heated at 100°C for different periods of time, and 17 protein bands/spots were positively identified by MALDI TOF/TOF MS/MS. Protein profiles on both 1-DE and 2-DE gels changed obviously and more polydisperse bands/spots were observed with increased heating time for over-heated samples. Based on these observations, an alternative protein marker-producing method was designed by directly dissolving protein standards without BSA into lysis buffer. This new kind of protein marker could be stored at room temperature for a long time, thus was more convenient for using and shipping. The identification of 17 proteins via MS and comparison of their identities revealed MASCOT-searched scores, number of both matched peptides, total searched peptides and sequence coverage became progressively lower with increasing denaturation intensity, probably due to the interference of thermal denaturation on trypsin cleavage efficiency and produced redundant modified peptides. Therefore, it was concluded that thermal denaturation not only changed the protein profiles and produced more polydisperse protein bands/spots, but also heavily interfered with the subsequent MS analysis, hence not recommended in future proteomic analysis for proteins dissolved in lysis buffer.     

A protein extraction method compatible with proteomic analysis for the euhalophyte Salicornia europaea

Protein extraction from plants like the halophyte Salicornia europaea has been problematic using standard protocols due to high concentrations of salt ions in their cells. We have developed an improved method for protein extraction from S. europaea, which allowed us to remove interfering compounds and salt ions by including the chemicals borax, polyvinylpolypyrrolidone, and phenol. The comparative study of this method with several other protocols using NaCl-treated S. europaea shoots demonstrated that this method gave the best distinction of proteins on 2-DE gels. This protocol had a wide range of applications as high yields and good distinction of 1-DE gels for proteins isolated from twelve other plants were rendered. In addition, we reported results of 2-DE using the recalcitrant tissue of the S. europaea roots. We also demonstrated that this protocol is compatible with proteomic analysis as eight specific proteins generated by this method have been identified by MS. In conclusion, our newly developed protein extraction protocol is expected to have excellent applications in proteomic studies of halophytes.     

Changes in amniotic fluid and umbilical cord serum proteomic profiles of foetuses with intrauterine growth retardation

Foetal growth is a result of a complex net of processes, requiring coordination within the maternal, placental, and foetal compartments, the imbalance or lack of which may lead to intrauterine growth restriction (IUGR). IUGR is the major cause of perinatal morbidity and mortality, and is also related to enhanced morbidity and metabolic abnormalities later in life. In the present study, the protein profiles of umbilical cord serum (UCS) and amniotic fluid (AF) of ten IUGR and ten appropriate for gestational age newborns have been analysed by 2-DE, and nanoHPLC-Chip/MS technology. A total of 18 and 13 spots were found to be differentially expressed (p<0.01) in UCS and AF respectively. The unique differentially expressed proteins identified by MS/MS analysis were 14 in UCS, and 11 in AF samples. Protein gene ontology classification indicate that 21% of proteins are involved in inflammatory response, 20% in immune response, while a smaller proportion are related to transport, blood pressure, and coagulation. These results support the conclusion that the IUGR condition alters the expression of proteins involved in the coagulation process, immune mechanisms, blood pressure and iron and copper homeostasis control, offering a new insight into IUGR pathogenesis.