Optimization of reversed-phase microcapillary liquid chromatography for quantitative proteomics

A specific capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS) method for the determination of serotonin (5HT) and its precursors tryptophan (Trp) and 5-hydroxytryptophan (5HTP) in human platelet rich plasma is described. The analytes were removed from the plasma samples and preconcentrated by solid phase extraction (SPE) on mixed mode cation-exchange sorbents. The SPE recoveries were 71.6 +/- 3.1 for 5HT, 91.0 +/- 2.8 for Trp, and 95.3 +/- 5.9% for 5HTP. Deuterated analogues of 5HT and Trp were used as internal standards for quantitation purposes. Submicromolar detection limits were obtained for standard mixtures of all compounds and their deuterated isotopes, except 5HTP, which had detection limits in the low micromolar range. The potential usefulness of this method in the clinical setting was demonstrated by analyzing plasma extracts from healthy volunteers as well as from pathological samples. While 5HTP was not present in any of the analyzed samples, the levels of 5HT and Trp in both normal and pathological plasma were determined.     

Identification of proteins binding to decursinol by chemical proteomics

Decursinol, found in the roots of Angelica gigas Nakai, has been traditionally used to treat anemia and other various diseases. Recently, numerous biological activities such as cytotoxic effect on leukemia cells, and antitumor, neuroprotection, and antibacterial activities have been reported for this compound. Although a number of proteins including protein kinase C, androgen receptor, and acetylcholinesterase were proposed as molecular targets responsible for the activities of decursinol, they are not enough to explain such a diverse biological activity mentioned above. In this study, we employed a chemical proteomic approach, leading to identification of seven proteins as potential proteins interacting with decursinol. Most of the proteins contain a defined ATP or nucleic acid binding domain and have been implied to be involved in the pathogenesis and progression of various human diseases including cancer, autoimmune disorders, or neurodegenerative diseases. The present results may provide clues to understand the molecular mechanism of the biological activities shown by decursinol, an anticancer natural product.     

Evaluation of comprehensive multidimensional separations using reversed-phase, reversed-phase liquid chromatography/mass spectrometry for shotgun proteomics

Two-dimensional liquid-chromatographic (LC) separation followed by mass spectrometric (MS) analysis was examined for the identification of peptides in complex mixtures as an alternative to widely used two-dimensional gel electrophoresis followed by MS analysis for use in proteomics. The present method involves the off-line coupling of a narrow-bore, polymer-based, reversed-phase column using an acetonitrile gradient in an alkaline mobile phase in the first dimension with octadecylsilanized silica (ODS)-based nano-LC/MS in the second dimension. After the first separation, successive fractions were acidified and dried off-line, then loaded on the second dimension column. Both columns separate peptides according to hydrophobicity under different pH conditions, but more peptides were identified than with the conventional technique for shotgun proteomics, that is, the combination of a strong cation exchange column with an ODS column, and the system was robust because no salts were included in the mobile phases. The suitability of the method for proteomics measurements was evaluated.     

Measurement of oxalate in human plasma ultrafiltrate by ion chromatography

An improved ion chromatographic method for the measurement of oxalate in human plasma ultrafiltrate is described. Ultrafiltration was carried out using an appropriate device and procedure. Centrifugation of 0.5 ml heparin plasma at 4°C for 50 min yielded water-clear ultrafiltrate in amounts allowing replicate measurements of oxalate. The specificity of the method was confirmed. The recovery of oxalate added to plasma was approximately 80%, whereas dilution of plasma, and of an oxalate-containing salt solution, resulted in falsely high values; the mechanism(s) underlying this phenomenon are insufficiently understood at present. The intra-assay of the method was assessed and from replicates of a pool plasma, the inter-assay precision from ten measurements of the same plasma on different days; the observed ranges of oxalate were 1.32-1.56 (mean 1.42) and 1.42-1.64 (mean 1.53) μmol/l, respectively. In plasma ultrafiltrate of a limited number of healthy volunteers the range of oxalate was 1.81-2.50 μmol/l, thus permitting renal oxalate handling to be studied.     

Hydrochloric acid hydrolysis of proteins and determination of tryptophan by reversed-phase high-performance liquid chromatography

A simple method was developed for determination of tryptophan in proteins. Hydrolysis is achieved under reducing conditions, in 6 N hydrochloric acid containing 0.4% beta-mercaptoethanol, at 110 degrees C for 24 h. The phenylthiocarbamyl derivatives of the amino acids are separated by reversed-phase high-performance liquid chromatography, without any by-product interference. The recovery of tryptophan is complete. However, the method does not allow the determination of tryptophan in carbohydrate-rich biological samples.     

Identification of parathyroid hormone-regulated proteins in mouse bone marrow cells by proteomics

The ability of parathyroid hormone (PTH) to enhance bone formation has recently been exploited in the treatment of osteoporosis. Several studies have suggested that the activation of bone marrow stromal cells could be preceded to show the anabolic effect of PTH on bone formation, but little is known of PTH-regulated proteins in bone marrow cells. Therefore, protein profiling in the intermittent PTH-treated bone marrow cells was evaluated using proteomics. Daily treatment for 5 days consisting of subcutaneous injection of either 150 microg/kg per day of mouse PTH (1-84) or vehicle (0.9% normal saline) was performed on the ICR mouse. At the end of the treatment period, bone marrow cells were separated and used in proteomics. The expression levels of seven proteins including vimentin were decreased, but those of four proteins including calreticulin and thioredoxin domain containing 7 protein (Txnde7) were increased. Among these, the decrease of vimentin and the increase of both calreticulin Txnde7 in mRNA levels were confirmed by semi-quantitative RT-PCR. In PTH-treated mouse MC3T3-E1 osteoblast cells, mRNA expression levels were not totally consistent with the results observed in proteomics. In conclusion, the differentially expressed proteins in bone marrow cells depending on PTH could be highly linked to the differentiation of osteoprogenitor cells in the bone marrow into preosteoblast cells.     

Identification of tumor-associated proteins in oral tongue squamous cell carcinoma by proteomics

Oral tongue carcinoma is an aggressive tumor that particularly affects chronic smokers, drinkers and betel squid chewers. Patients often present symptoms at a late stage, and there is a high recurrence rate after treatment. In this article, we report the first proteomic analysis of oral tongue carcinoma to globally search for tumor related proteins. Apart from helping us to understand the molecular pathogenesis of the carcinoma, these proteins may also have potential clinical applications as biomarkers, enabling the tumor to be identified at an early stage in high risk individuals, treatment response to be predicted, and residual or recurrent carcinoma to be detected sooner after treatment. The protein expression profiles of ten oral tongue squamous cell carcinomas and their matched normal mucosal resection margins were examined by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectroscopy. A number of tumor-associated proteins including heat shock protein (HSP)60, HSP27, alpha B-crystalline, ATP synthase beta, calgranulin B, myosin, tropomyosin and galectin 1 were consistently found to be significantly altered in their expression levels in tongue carcinoma tissues, compared with their paired normal mucosae. The expression profile portrays a global protein alteration that appears specific to oral tongue cancer. The potential of utilizing these tumor related proteins for screening cancer and monitoring recurrence warrants further investigation.     

Identification of proteins involved in osmotic stress response in Enterobacter sakazakii by proteomics

Enterobacter sakazakii is considered an opportunistic food-borne pathogen, causing rare but significant illness especially in neonates. It has been proposed that the organism is relatively resistant to osmotic and dry stress compared to other species of the Enterobacteriaceae group. To understand the mechanisms involved in osmotic stress response, 2-DE protein analysis coupled to MALDI-TOF MS was employed to investigate changes in the protein profiles of E. sakazakii cells in response to two different types of osmotic stress (physical desiccation and growth in hyperosmotic media). In total, 80 differentially expressed protein spots corresponding to 53 different protein species were identified. Affiliation of proteins to functional categories revealed that a considerable number of the differentially expressed proteins from desiccated and hyperosmotic grown samples belonged to the same functional category but were regulated in opposite directions. Our data show that the protein pattern of NaCl-grown cultures reflect more or less a general down-regulation of central metabolic pathways, whereas adaptation of (non-growing) cells in a desiccated state represents an accumulation of proteins that serve some structural or protective role. The most striking effects observed for both types of osmotic stress in E. sakazakii were a significant down-regulation of the motility apparatus and the formation of filamentous cells.     

Identification of human nasal mucous proteins using proteomics

The determination of possible biomarkers in nasal secretion of healthy subjects can have a role in early diagnosis of diseases such as rhinosinusitis. For this purpose, nasal lavage fluids (NLFs) from ten volunteers, collected before and after they had been submitted to nasal provocations, were investigated. Separation and analysis of proteins present in this complex matrix was performed using a capillary liquid chromatography-electrospray-quadrupole-time of flight mass spectrometry equipment. From among a total of 111 proteins found (89 known and two unknown proteins), 42 of which had never been previously described in this fluid, such as Deleted in Malignant Brain Tumors 1 isoform a precursors, and cytoskeletal proteins were identified with high statistical score. Three proteins of palate lung nasal epithelial clone (PLUNC) family: SPLUNC1, LPLUNC1, and LPLUNC2 were identified. Proteins involved in innate (27%) and acquired immunity (21%) systems were major components of NLF. Cellular (52% of all proteins identified) such as cytoskeletal (33%), functional (15%), and regulatory (4%) proteins, normally present in the nasal cavity, have also been identified. The proteomic approach presented here allowed us to identify the proteins involved in acquired and innate immune response in the nose against microbial infections and unclean inhaled air.     

Identification of novel surface proteins of Anaplasma phagocytophilum by affinity purification and proteomics

Anaplasma phagocytophilum is the etiologic agent of human granulocytic anaplasmosis (HGA), one of the major tick-borne zoonoses in the United States. The surface of A. phagocytophilum plays a crucial role in subverting the hostile host cell environment. However, except for the P44/Msp2 outer membrane protein family, the surface components of A. phagocytophilum are largely unknown. To identify the major surface proteins of A. phagocytophilum, a membrane-impermeable, cleavable biotin reagent, sulfosuccinimidyl-2-[biotinamido]ethyl-1,3-dithiopropionate (Sulfo-NHS-SS-Biotin), was used to label intact bacteria. The biotinylated bacterial surface proteins were isolated by streptavidin agarose affinity purification and then separated by electrophoresis, followed by capillary liquid chromatography-nanospray tandem mass spectrometry analysis. Among the major proteins captured by affinity purification were five A. phagocytophilum proteins, Omp85, hypothetical proteins APH_0404 (designated Asp62) and APH_0405 (designated Asp55), P44 family proteins, and Omp-1A. The surface exposure of Asp62 and Asp55 was verified by immunofluorescence microscopy. Recombinant Asp62 and Asp55 proteins were recognized by an HGA patient serum. Anti-Asp62 and anti-Asp55 peptide sera partially neutralized A. phagocytophilum infection of HL-60 cells in vitro. We found that the Asp62 and Asp55 genes were cotranscribed and conserved among members of the family Anaplasmataceae. With the exception of P44-18, all of the proteins were newly revealed major surface-exposed proteins whose study should facilitate understanding the interaction between A. phagocytophilum and the host. These proteins may serve as targets for development of chemotherapy, diagnostics, and vaccines.     

Desalting and concentration of proteins in dilute solution using reversed-phase high-performance liquid chromatography

A rapid method for desalting and concentrating dilute protein solutions using short reversed-phase columns (3-4 cm) has been described. The recovery of proteins is usually 90-100%. The method is simple and rapid and allows the desalting and concentration of protein samples simultaneously. A wide variety of proteins in the range up to 80 kDa can be desalted in microgram to milligram amounts, and volumes up to 1 liter can be concentrated to a few milliliters by a single injection.     

Immunodepletion of high abundance proteins coupled on-line with reversed-phase liquid chromatography: A two-dimensional LC sample enrichment and fractionation technique for mammalian proteomics

Proteomic analysis can be hampered by the large concentration distribution of proteins. Immunoaffinity techniques have been applied to selectively remove high abundant proteins (HAP's) from samples prior to analysis. Although immunodepletion of HAP's has been shown to enable greater detection of low abundance proteins, the resulting fractions are often diluted 5–10-fold during the process. Various concentration techniques can be applied; however, many are incompatible with the high salt content of the fractions. To help overcome this limitation, a two-dimensional liquid chromatography (2D-LC) method was developed which couples an IgY immunodepletion column in the first dimension with a large pore C18 analytical column in the second. A protein trap cartridge serves as an injection loop between the columns to facilitate on-line concentration and desalting. Feasibility of this 2D-LC system was demonstrated for mammalian proteomics. Beyond depletion of interfering proteins, this instrumentation provides four advantages which make immunodepletion technology more convenient, including: (1) on-line desalting (2) automatic buffer exchange (3) facile concentration and (4) fractionation by polarity.     

Identification of paracrine neuroprotective candidate proteins by a functional assay-driven proteomics approach

Glial cells support neuronal survival and function by secreting neurotrophic cytokines. Retinal Mueller glial cells (RMGs) support retinal neurons, especially photoreceptors. These highly light-sensitive sensory neurons receive vision, and their death results in blinding diseases. It has been proposed that RMGs release factors that support photoreceptor survival, but the nature of these factors remains to be elucidated. To discover such neurotrophic factors, we developed an integrated work flow toward systematic identification of neuroprotective proteins, which are, like most cytokines, expressed only in minute amounts. This strategy can be generally applied to identify secreted bioactive molecules from any body fluid once a recipient cell for this activity is known. Toward this goal we first isolated conditioned medium (CM) from primary porcine RMGs cultured in vitro and tested for survival-promoting activity using primary photoreceptors. We then developed a large scale, microplate-based cellular high content assay that allows rapid assessment of primary photoreceptor survival concomitant with biological activity in vitro. The enrichment strategy of bioactive proteins toward their identification consists of several fractionation steps combined with tests for biological function. Here we combined 1) size fractionation, 2) ion exchange chromatography, 3) reverse phase liquid chromatography, and 4) mass spectrometry (Q-TOF MS/MS or MALDI MS/MS) for protein identification. As a result of this integrated work flow, the insulin-like growth factor-binding proteins IGFBP5 and IGFBP7 and connective tissue growth factor (CTGF) were identified as likely candidates. Cloning and stable expression of these three candidate factors in HEK293 cells produced conditioned medium enriched for either one of the factors. IGFBP5 and CTGF, but not IGFBP7, significantly increased photoreceptor survival when secreted from HEK293 cells and when added to the original RMG-CM. This indicates that the survival-promoting activity in RMG-CM is multifactorial with IGFBP5 and CTGF as an integral part of this activity.     

Sequential sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reversed-phase chromatography of unfolded proteins

Sequential sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and reversed-phase high performance liquid chromatography on a fluorocarbon packing (poly F column, DuPont) provide fully denatured but highly purified protein, which is free of low-molecular-weight substances and directly amenable to structural analysis. Conditions for the preparative elution of four test proteins (bovine serum albumin, carbonic anhydrase, myoglobin, cytochrome c) blotted to Immobilon membranes have been optimized. Phenol saturated with 50 mM Tris-HCl (pH 8.25) and containing 2% SDS and 1% 2-mercaptoethanol is able to elute proteins that have been blotted to Immobilon membranes, stained with Coomassie R-250, and stored as dry sheets, largely irrespective of their molecular mass. Polypeptides that are not degraded by exposure to low pH can also be efficiently extracted directly from stained gels with 70% formic acid. If further separation of polypeptides is not needed, a simple run of a protein sample dissolved in 1-2% SDS on the poly F column will efficiently remove low-molecular-weight substances, including SDS.     

Determination of albumin and myoglobin in dialysate and ultrafiltrate samples by high-performance size-exclusion chromatography

A high-performance size-exclusion chromatographic method was developed, validated and implemented for simultaneous and quantitative determination of albumin and myoglobin along with inulin, vancomycin and creatinine in dialysate and ultrafiltrate samples from in vitro hemodialysis experiments. The experimental parameters including mobile phase pH, ionic strength, detection wavelength, flow-rate, injection volume were first optimized for the determination of albumin, myoglobin, inulin, vancomycin and creatinine. The peak height ratio and detection limits of the proteins were then comparatively studied at 210, 254 and 280 nm by UV and diode array detection. The method was further validated by evaluating the linearity, precision and accuracy of the proteins. The assay was finally implemented to the simultaneous and quantitative determination of the proteins in dialysate and ultrafiltrate samples.     

Identification of differentially expressed proteins in colorectal cancer by proteomics: down-regulation of secretagogin

To identify proteins with colorectal cancer-specific regulation, comparative 2-DE of individual-matched normal and neoplastic colorectal tissue specimens was performed. We found 15 protein spots with concordantly increased and 20 protein spots with concordantly decreased intensity in tumor tissue (expression regulation more than fivefold). Nine of these proteins were identified by MS/MS. Interestingly, one of the proteins, which exhibited a marked down-regulation in colorectal cancer tissues, was the recently identified endocrine cell-expressed protein secretagogin. The reduction of the secretagogin content in colorectal cancer tissues was confirmed by comparative immunoblotting (n = 17) and RT-PCR (n = 22) as well as by immunohistochemistry (n = 45) of individual-matched neoplastic and normal colorectal tissue specimens. Immunohistochemistry revealed absence of secretagogin-expressing cells in most of the colorectal cancer tissue specimens. However, some colorectal cancers were characterized by secretagogin-expressing cells. In normal mucosa, positively stained cells exhibited a neuroendocrine cell-characteristic morphology and mucosal location. In colorectal cancer tissues, secretagogin-expressing cells were characterized by a malignant morphology. Our findings might represent the basis for the clinical application of secretagogin as a biomarker for a distinct subgroup of colorectal cancers.