Serological comparative proteomics analysis of mitochondrial autoantibody‐negative and ‐positive primary biliary cirrhosis

Here, we investigated the pathogenesis of primary biliary cirrhosis (PBC) by using 2D‐DIGE to analyze serological differences between anti‐mitochondrial antibody (AMA)‐positive and ‐negative PBC patients. The study comprised 30 patients with PBC; 20 AMA‐positive and ten AMA‐negative patients matched for age, sex , and pathological stage. A screening group (four AMA‐positive and four AMA‐negative patients) was used for 2D‐DIGE. Protein spots that were differently abundant between the two groups were identified via dye intensity and MS. Nine candidate proteins were identified from these spots. Western blotting was used to verify two of the identified proteins, serum amyloid P‐component (SAP) and vitronectin (VN). VN levels were significantly higher in the sera of AMA‐negative PBC patients (p < 0.01), whereas no significant difference was found between the two groups for SAP. To our knowledge, this is the first study to use serological comparative proteomics to explore differences between AMA‐positive and ‐negative PBC patients. VN levels were higher in AMA‐negative PBC patients, and this finding could be related to the more severe bile duct destruction observed in this group.     

Improved consistency in 2D gel electrophoresis: Sheep plasma as a test case

Two‐dimensional (2D) gel electrophoresis is a well‐proven proteomic technique; however, sample‐specific optimisation can often be necessary in order to get consistent quantitation. In particular, plasma samples are often smeared on 2D gels making spot matching difficult. A variety of different sample preparation and 2D methods were tested by using sheep plasma, and it was found that lowering sample pH prior to precipitation, using a long voltage gradient for isoelectric focusing and the inclusion of carrier ampholytes in the electrode wicks, improved both the quality and consistency of spot resolution. Analysis of the internal standards from two different DIGE experiments, one with conventional methodology and one with the improved method, showed that along with substantially improving the number of spots resolved, the average CV (coefficient of variation) of matched standards was lower with the new method. 428 matched spots were found using the improved method compared to 208 matched spots using conventional methodology. For the 174 spots that were matched between the two DIGE experiments, the average CV's of spot volumes were also significantly lower, at 0.20 for the new method compared to 0.24 for the conventional method (p < 0.001).     

Biomarker discovery in breast cancer serum using 2-D differential gel electrophoresis/ MALDI-TOF/TOF and data validation by routine clinical assays

In the present study, we used 2-D differential gel electrophoresis (2-D DIGE) and MS to screen biomarker candidates in serum samples obtained from 39 patients with breast cancer and 35 controls. First, we pooled the serum samples matched with age and menopausal status. Then, we depleted the two most abundant proteins albumin and IgG by immunoaffinity chromatography under partly denaturing conditions in order to enrich low-abundance proteins and proteins with low molecular weight. Concentrated and desalted samples were labeled with three different CyDyes including one internal standard, pooled from all the samples, and separated with 2-D DIGE in triplicate experiments. Biological variations of the protein expression level were analyzed with DeCyder software and evaluated for reproducibility and statistical significance. The profile of differentially expressed protein spots between patients and controls revealed proapolipoprotein A-I, transferrin, and hemoglobin as up-regulated and three spots, apolipoprotein A-I, apolipoprotein C-III, and haptoglobin alpha2 as down-regulated in patients. Finally, routine clinical immunochemical reactions were used to validate selected candidate biomarkers by quantitative determination of specific proteins in all individual serum samples. The serum level of transferrin correlated well with the 2-D-DIGE results. However, the serum levels of apolipoprotein A-I and haptoglobin could not be detected with the clinical routine diagnostic tests. This demonstrated an advantage 2-D DIGE still has over other techniques. 2-D DIGE can distinguish between isoforms of proteins, where the overall immunochemical quantification does fail due to a lack of isoform-special antibodies.     

Comparison of protein precipitation methods from adipose tissue using difference gel electrophoresis

Proteomic methods have great potential to aid our understanding of the functional and pathological roles of adipose tissue. A critical initial step in the proteomic studies is the efficient isolation of proteins before conducting detailed analysis. In this study, three different methods were used for precipitating proteins; we analyzed samples from visceral adipose tissue, subcutaneous adipose tissue, and stromal visceral fraction extracts after chloroform/methanol, acetone, and trichloroacetic acid precipitation. The proteins recovered after the precipitation steps were examined by 2D‐DIGE. Statistical analyses were carried out using simple linear regression analyses and R2 values were calculated for the intra‐ and inter‐method comparisons. We found that all three precipitation methods provided highly reproducible protein spots that were recovered when run in duplicate using the same method of precipitation, irrespective of whether it was solvent (R2 = 0.85–0.98) or acid‐based (R2 = 0.80–0.96). A higher variation and poor correlation was noted for the recovered protein spots when comparisons were made between the methods (R2 = 0.40–0.88) and also when the same method was compared between different sample types. In this study, TCA‐precipitated samples were enriched in lower molecular mass proteins compared to the samples extracted by solvent‐based precipitation methods.     

Alpha‐1 antitrypsin variants in plasma from HIV‐infected patients revealed by proteomic and glycoproteomic analysis

Novel tools are necessary to explore proteins related to human immunodeficiency virus (HIV) infection. In this work, proteomic and glycoproteomic technology were employed to examine plasma samples from HIV‐positive patients. Through comparative proteome analysis of normal and HIV‐positive plasma samples, 19 differentially expressed protein spots related to 12 non‐redundant proteins were identified by ESI‐ion trap MS. Among these, the 130‐kDa isoform of α‐1‐antitrypsin was found to be decreased in HIV‐positive patients while another variant with a molecular weight of 40 kDa was increased. SWISS‐2‐D‐PAGE reference gel and protein sequence comparisons of the 40‐kDa protein showed homology with α‐1‐antitrypsin minus the N‐terminus, and its identity was further confirmed by 1‐D Western blotting and glycoproteomic analysis. In all, our results showed that proteomics and glycoproteomics are powerful tools for discovering proteins related to HIV infection. Furthermore, this 40‐kDa variant of α‐1‐antitrypsin found in the plasma of HIV‐positive individuals may prove to be a potentially useful biomarker for anti‐HIV research according to bioinformatics analysis.     

Metabolic Profiling of Human Colorectal Cancer Using High Resolution 1H Nuclear Magnetic Resonance Spectroscopy

Colorectal cancer (CRC) is the third commonest malignancy cancer worldwide. Clear understandings of global metabolic profiling of the normal mucosa and cancer tissues are vitally important to aid optimizing the clinical management strategy and understanding CRC biology. We studied metabolic characteristics of 20 CRC and 20 distant normal mucosa tissues extracts from 20 patients using high resolution ¹H NMR spectroscopy in conjunction with multivariate analyses, such as principal component analysis (PCA). Compared with distant normal mucosa tissues, lactate, taurine, ornithine and polyamine were present at significantly higher levels in CRC tissue extracts whereas myo‐inositol was present at significantly lower level. Two metabolites ratios such as myo‐inositol/taurine and myo‐inositol/(ornithine+polyamine) appear to be the most valuable biomarkers for the differentiation CRC from normal mucosa tissues. Our data suggested that HR ¹H NMR spectroscopy combined with multivariate analyses is a potentially useful technology for detecting malignant changes in the normal mucosa tissues, the technique may be further exploited for future CRC biomarker research or identification of targets for therapeutic manipulations.     

2D difference gel electrophoresis reference map of a Fusarium graminearum nivalenol producing strain

Fusarium graminearum is widely studied as a model for toxin production among plant pathogenic fungi. A 2D DIGE reference map for the nivalenol‐producing strain 453 was established. Based on a whole protein extract, all reproducible spots were systematically picked and analyzed by MALDI‐TOF/TOF, leading to the identification of 1102 protein species. The obtained map contributes to the annotation of the genome by identifying previously nondescribed hypothetical proteins and will serve as a reference for future studies aiming at deciphering F. graminearum biology and chemotype diversity.     

High-performance liquid chromatography/nano-electrospray ionization tandem mass spectrometry, two-dimensional difference in-gel electrophoresis and gene microarray identification of lymphatic metastasis-associated biomarkers

The potential biomarkers for the lymphatic metastatic process of mouse hepatocarcinoma were investigated by using two-dimensional difference in-gel electrophoresis (2D DIGE), high-performance liquid chromatography/nano-electrospray ionization tandem mass spectrometry (HPLC/nESI-MS/MS) and GeneChip. 2D DIGE was performed to screen and quantify the differentially expressed proteins between two well-established mouse hepatocarcinoma cell lines, Hca-F with 75% and Hca-P with 25% metastasis rate of lymph node potentials. The protein spots in the gel were visualized by the highly sensitive Deep Purple (GE Healthcare) fluorescent stain. Protein identification was obtained for gel spots by HPLC/nESI-MS/MS analysis with high quality. GeneChip microarray was performed to identify genes differentially expressed at the mRNA level. Seventeen genes including the chloride intracellular channel l, caspase 3, fructose bisphosphatase 2, glutamate dehydrogenase 1, V-crk sarcoma virus CT10 oncogene homolog, N-myc downstream regulated gene1, villin2, gelsolin, enoyl coenzyme A hydratase 1, transketolase, vimentin, annexins A5 and A7, keratin complex2 basic gene7 and gene8, lactamase (bata 2) and Ero1-like protein were found abnormally regulated and expressed concordantly both at the protein and mRNA levels between the two cell lines. More than half of these genes were for the first time revealed to be involved directly in hepatocarcinoma due to the lymphatic metastasis. The interdisciplinary combination of HPLC/nESI-MS/MS with 2D DIGE and GeneChip techniques opens up the possibility for the biomarker discovery of disease with high confidence.     

Size‐matched alkyne‐conjugated cyanine fluorophores to identify differences in protein glycosylation

Currently, there are few methods to detect differences in posttranslational modifications (PTMs) in a specific manner from complex mixtures. Thus, we developed an approach that combines the sensitivity and specificity of click chemistry with the resolution capabilities of 2D‐DIGE. In “Click‐DIGE”, posttranslationally modified proteins are metabolically labeled with azido‐substrate analogs, then size‐ and charge‐matched alkyne‐Cy3 or alkyne‐Cy5 dyes are covalently attached to the azide of the PTM by click chemistry. The fluorescently‐tagged protein samples are then multiplexed for 2DE analysis. Whereas standard DIGE labels all proteins, Click‐DIGE focuses the analysis of protein differences to a targeted subset of posttranslationally modified proteins within a complex sample (i.e. specific labeling and analysis of azido glycoproteins within a cell lysate). Our data indicate that (i) Click‐DIGE specifically labels azido proteins, (ii) the resulting Cy‐protein conjugates are spectrally distinct, and (iii) the conjugates are size‐ and charge‐matched at the level of 2DE. We demonstrate the utility of this approach by detecting multiple differentially expressed glycoproteins between a mutant cell line defective in UDP‐galactose transport and the parental cell line. We anticipate that the diversity of azido substrates already available will enable Click‐DIGE to be compatible with analysis of a wide range of PTMs.     

Serum metabolomics as a novel diagnostic approach for gastrointestinal cancer

Conventional tumor markers are unsuitable for detecting carcinoma at an early stage and lack clinical efficacy and utility. In this study, we attempted to investigate the differences in serum metabolite profiles of gastrointestinal cancers and healthy volunteers using a metabolomic approach and searched for sensitive and specific metabolomic biomarker candidates. Human serum samples were obtained esophageal (n = 15), gastric (n = 11), and colorectal (n = 12) cancer patients and healthy volunteers (n = 12). A model for evaluating metabolomic biomarker candidates was constructed using multiple classification analysis, and the results were assessed with receiver operating characteristic curves. Among the 58 metabolites, the levels of nine, five and 12 metabolites were significantly changed in the esophageal, gastric and colorectal cancer patients, respectively, compared with the healthy volunteers. Multiple classification analysis revealed that the variations in the levels of malonic acid and l ‐serine largely contributed to the separation of esophageal cancer; gastric cancer was characterized by changes in the levels of 3‐hydroxypropionic acid and pyruvic acid; and l ‐alanine, glucuronoic lactone and l ‐glutamine contributed to the separation of colorectal cancer. Our approach revealed that some metabolites are more sensitive for detecting gastrointestinal cancer than conventional biomarkers. Our study supports the potential of metabolomics as an early diagnostic tool for cancer. Copyright © 2011 John Wiley & Sons, Ltd.     

Microscale solution IEF combined with 2-D DIGE substantially enhances analysis depth of complex proteomes such as mammalian cell and tissue extracts

Current gel-based protein profiling methods such as 2-DE and fluorescent 2-D difference in gel electrophoresis (DIGE) evaluate small portions of complex proteomes. Hence, sample prefractionation is essential for more comprehensive proteome coverage and detection of low-abundant proteins. In this study, we describe the combination of DIGE labeling with microscale solution IEF (MicroSol-IEF) fractionation and subsequent analysis on slightly overlapping narrow pH range 2-D gels. By fluorescently tagging and mixing samples and controls prior to prefractionation, complications resulting from minor run-to-run variations during MicroSol-IEF separations of multiple samples are avoided. This greatly improves the reliability of quantitative comparisons. To illustrate its utility, this 3-D DIGE strategy was applied to analysis of human melanoma cells and mouse lung tissue extracts. Approximately 1000 reproducible spots can be obtained from narrow range 2-D gels of individual MicroSol-IEF fractions, and approximately 6000 spots can be obtained from entire proteomes. Quantitative changes in closely related samples could be more reliably detected and the method has a greatly increased capacity to distinguish between closely related protein isoforms. Thus the 3-D DIGE strategy produces a powerful method for more comprehensive and more reliable quantitative comparisons of protein profiles of very complex proteomes.     

Noninvasive detection of colorectal cancer by analysis of exhaled breath

There has been growing interest in exhaled breath analysis for cancer screening and disease monitoring; however, limited breath biomarker information exists regarding colorectal cancer (CRC). The objective of this study was to screen for breath biomarkers of CRC. Exhaled breath was collected from 20 CRC patients and 20 healthy controls; subsequently, solid-phase microextraction–gas chromatography/mass spectrometry (SPME-GC/MS) was used to assess the exhaled volatile organic compounds (VOCs) of the study participants. The statistical methods of principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were performed to process the final data. The VOCs in the exhalations of CRC patients exhibited significant differences from the VOCs in the exhalations of healthy controls; in particular, relative to the latter exhalations, the former exhalations contain significantly higher levels of cyclohexanone, 2,2-dimethyldecane, dodecane, 4-ethyl-1-octyn-3-ol, ethylaniline, cyclooctylmethanol, trans-2-dodecen-1-ol, and 3-hydroxy-2,4,4-trimethylpentyl 2-methylpropanoate but significantly lower levels of 6-t-butyl-2,2,9,9-tetramethyl-3,5-decadien-7-yne (P?<?0.05). Analyses of breath VOCs provide a related model of CRC exhalation that could represent an effective and convenient screening method for this disease.

Setup for human sera MALDI profiling: the case of rhEPO treatment

The implementation of high-throughput technologies based on qualitative and quantitative methodologies for the characterization of complex protein mixtures is increasingly required in clinical laboratories. MALDI profiling is a robust and sensitive technology although the serum high dynamic range imposes a major limitation hampering the identification of less abundant species decreasing the quality of MALDI profiling. A setup to improve these parameters has been performed for recombinant human erythropoietin (rhEPO) monitoring in serum, analyzing the effects of two commercially available columns (MARS Hu7 and Hu14) for immunodepletion, and two matrices (α-cyano-4-hydroxycinnamic acid and 2',4'-dihydroxyacetophenone) for peak quality improvement. The immunodepletion capability of both columns was determined by 2-D DIGE, which precisely revealed the efficacy of Hu14 in protein removal and the serum dynamic range decrement. In addition, the type of matrix, the sample dilution, and the efficacy of optimized parameters were used for serum profiling of ten healthy subjects before and after rhEPO treatment. The principal component analysis indicates that a combination of Hu14 column and 2',4'-dihydroxyacetophenone matrix increases data quality allowing the discrimination between treated and untreated samples, making serum MALDI profiling suitable for clinical monitoring of rhEPO.     

Horizontal comparative fluorescence two‐dimensional gel electrophoresis for improved spot coordinate detection

Vertical comparative 2D fluorescence gel electrophoresis (CoFGE) has recently been shown to increase the reproducibility of coordinate assignment for protein spots, in particular in singular experiments, which cannot be investigated using DIGE. The method applies a standardized marker grid formed by a set of purified proteins to the sample proteome in a conglomerate of 1DE, 2DE, and DIGE. Here, improvements are demonstrated by transferring CoFGE to horizontal 2DE. These include the elimination of the protein modification by residual acrylamide monomer unavoidable in vertical CoFGE, reduced buffer volumes, and highly efficient laboratory procedures. Spot patterns are well defined and can be easily analyzed using commercially available warping algorithms. With horizontal CoFGE also a correction for changes in pI was introduced using a third fluorescent dye. Horizontal CoFGE holds high promises in comparative proteomics.     

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.     

Exploring 9‐arylcarbazole moiety as the building block for the synthesis of photoluminescent group 10–12 heavy metal diynes and polyynes with high‐energy triplet states

A new series of organic‐soluble and thermally stable group 10 platinum(II) polyyne polymers functionalized with 9‐arylcarbazole moiety trans‐[? Pt(PBu₃)₂C?CRC?C? ]_n (R = 9‐arylcarbazole‐3,6‐diyl; aryl = phenyl, p‐methylphenyl, p‐fluorophenyl) were prepared in good yields by Hagihara's dehydrohalogenative polymerization of trans‐[PtCl₂(PBu₃)₂] with HC?CRC?CH under ambient conditions. The regiochemical structures of the polymers were characterized by multinuclear NMR spectroscopy. We discuss the optical spectroscopy of these polymetallaynes and compare the results with their bimetallic molecular model complexes trans‐[Pt(Ph)(PEt₃)₂C?CRC?CPt(Ph)(PEt₃)₂] as well as its group 11 gold(I) and group 12 mercury(II) congeners [(PPh₃)AuC?CRC?CAu(PPh₃)] and [MeHgC?CRC?CHgMe]. The structural properties of several model complexes were studied by X‐ray crystallography. The influence of the heavy metal atom and the 9‐aryl substituent of carbazole on the phosphorescence behavior and the spatial distribution of the lowest singlet (S₁) and triplet (T₁) excitons in these metalated alkynyl systems are comprehensively elucidated. The present work indicates that the efficiency of organic triplet emissions harnessed through the heavy‐atom effect of group 10–12 transition metals in the main chain generally follows the order Pt > Au > Hg but the optical properties of the materials are relatively insensitive to the nature of the 9‐aryl group on the carbazolyl ring. All of these metallaynyl‐carbazole materials with high‐energy T₁ states of 2.68 eV or higher show high phosphorescence efficiencies at low temperatures. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 5588–5607, 2006     

Comparative proteomic analysis of different Toxoplasma gondii genotypes by two‐dimensional fluorescence difference gel electrophoresis combined with mass spectrometry

Toxoplasma gondii is a protozoan parasite infecting almost all warm‐blooded animals and humans. There are three infective stages of T. gondii: the tachyzoites, the bradyzoites, and the oocysts. The tachyzoite is a rapidly multiplying stage and the main pathogenic factor. In North America and Europe, T. gondii is consisted of four major clonal lineages (namely Types I, II, III, and Type 12). In this study, we explored the proteomic profiles of different genotypes (Type I‐RH strain, Type II‐PRU strain, Type II‐TgQHO strain, and ToxoDB 9‐TgC7 strain) of T. gondii tachyzoites by using 2D DIGE combined with MALDI‐TOF MS. Totally, 110 differentially abundant protein spots were selected. Of these, 98 spots corresponding to 56 proteins from T. gondii were successfully identified. These included surface antigen (SAG1), heat shock protein 70 (Hsp 70), disulfide isomerase, coronin, heat shock protein 60 (Hsp 60), pyruvate kinase, receptor for activated C kinase 1, and peroxiredoxin. Gene ontology enrichment analysis revealed that most of the differentially abundant proteins were involved in biological regulation, metabolic process, response to stress, binding, antioxidant activity, and transporter activity. According to the KEGG metabolic pathway maps of T. gondii, some identified proteins were involved in the glycolytic/gluconeogenesis pathway. The present study identified differentially abundant proteins among different genotypes of T. gondii and these findings have implications for the better understanding of the phenotypic differences among the examined T. gondii genotypes, which in turn may contribute to the better control of toxoplasmosis.     

Synthesis and light‐emitting properties of platinum‐containing oligoynes and polyynes derived from oligo(fluorenyleneethynylenesilylene)s

A series of blue‐light‐emitting oligo(fluorenyleneethynylenesilylene)s (OFESs) of the general formula HC?CRC?C(EC?CRC?C)_mEC?CRC?CH (E = SiPh₂, SiMe₂, or SiMe₂? SiMe₂; m = 0–2; R = 9,9‐dihexylfluorene‐2,7‐diyl) and their phosphorescent platinum‐containing oligoynes and polyynes were synthesized and characterized. The solution properties and regiochemical structures of this new structural class of organosilicon‐based polyplatinayne polymers {trans‐[? Pt(PBu₃)₂C ?CRC?C(EC?CRC?C)_mEC?CRC?C? ]_n} were studied with IR and NMR (¹H, ¹³C, ²⁹Si, and ³¹P) spectroscopy. The optical absorption and photoluminescence spectra of these metallopolymers were examined and compared with their discrete oligomeric model complexes. Our studies led to a novel approach of using the sp³‐silyl moiety as a conjugation interrupter to limit the effective conjugation length in metal polyynes, which could boost the phosphorescence decay rates essential for light‐energy harvesting from the triplet excited state. The influence of the heavy platinum atom and the group 14 silyl unit possessing different side‐group substituents on the thermal and phosphorescence properties was investigated in detail. We also established the goal of studying the evolution of the lowest singlet and triplet excited states with chain length m of OFESs and the nature of E in these metallopolymers. This work indicated that the phosphorescence emission efficiency harnessed through the heavy‐atom effect of platinum in the main chain did not change very much with oligomer chain length m but generally decreased with the E group in the order SiMe₂ > SiMe₂? SiMe₂ > SiPh₂. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 4804–4824, 2006     

Staining of proteins for 2D SDS‐PAGE using Coomassie Blue—speed versus sensitivity?

Several new fast staining protocols for the visualization of proteins separated by SDS‐PAGE utilizing Coomassie Blue staining (CBS) have been described in literature. The sensitivity of a newly designed staining protocol is usually estimated using 1D SDS‐PAGE of serially diluted protein samples. However, this approach is not predictive and satisfactory for 2D SDS‐PAGE capable of resolving hundreds or thousands of different proteins in a single analysis. In this work, a new fast staining protocol recently introduced by Dong et al. (PLoS One 2011, 6, e22394) was compared to colloidal CBS. The number of detectable spots in 2D SDS‐PAGE of identical blood plasma samples in repeated runs was chosen as a sensitivity criterion. Further, the influence of gel boiling on the subsequent protein identification by MS was investigated. In spite of its advantages, the staining protocol according to Dong et al. (PLoS One 2011, 6, e22394) seems to be less sensitive than colloidal Coomassie staining when the number of detected spots is the evaluating criterion. No obvious influence of gel boiling on the protein identification was observed.     

Determination of proline in human serum by a robust LC‐MS/MS method: application to identification of human metabolites as candidate biomarkers for esophageal cancer early detection and risk stratification

Altered serum proline levels are related to cancer metabolism. This study developed and validated a LC‐MS/MS method to analyze proline in human serum. Surrogate blank serum, coupled with stable isotope l ‐proline‐¹³C₅,¹⁵ N as internal standard, was used for generating standard curves ranging from 2.5 to 100 μg/mL. Proline was extracted from serum samples using methanol. A Phenomenex Lux 5u Cellulose‐1 column (250 × 4.6 mm) was used for chromatographic separation with 40% methanol in 0.05% formic acid aqueous solution as a mobile phase. Mass detection was performed under positive ionization electrospray. Intra‐ and inter‐day accuracy and precision were <10%. The extraction recovery and matrix factor were 99.17 and 1.47%, respectively. Our study showed that the chiral column had high specificity and selectivity for separating proline from serum components. The assay was successfully applied for the quantification of human serum proline levels from 30 esophageal cancer patients and 30 healthy volunteers. Statistical analyses showed significantly lower levels of serum proline in the patients as compared with the healthy volunteers (p‐value = 0.011). We report here a simple, specific and reproducible LC‐MS/MS method for the quantification of proline in human serum as a potential screening biomarker for esophageal cancer. Copyright © 2014 John Wiley & Sons, Ltd.