Low molecular weight proteins in urines from healthy subjects as well as diabetic,nephropathic and diabetic‐nephropathic patients: a MALDI study

Urine samples from healthy subjects as well as diabetic, nephropathic and diabetic‐nephropathic patients were analyzed by matrix assisted laser desorption/ionization (MALDI) mass spectrometry in order to establish evidence of some possible differences in the peptide profile related to the pathological states. Multivariate analysis suggested the possibility of a distinction among the considered groups of patients. Some differences have been found, in particular, in the relative abundances of three ions at m/z 1912, 1219 and 2049. For these reasons, further investigation was carried out by MALDI/TOF/TOF to determine the sequence of these peptides and, consequently, to individuate their possible origin. By this approach, the peptide at m/z 1912 was found to originate from uromodulin, and its lower expression in the case of nephropathy can be well related to the pathological condition. Ions at m/z 2049 and 1219 originate from the collagen α‐1(I) chain precursor and from the collagen α‐5 (IV) chain precursor, respectively, and, also in this case, their different expressions can be related to the pathologies under investigation. The obtained data seem to indicate that urine is an interesting biological fluid to investigate on the peptide profile and to obtain, consequently, information on the dismetabolism activated by specific pathologies. Copyright © 2009 John Wiley & Sons, Ltd.     

Comprehensive analysis of glycated human serum albumin tryptic peptides by off-line liquid chromatography followed by MALDI analysis on a time-of-flight/curved field reflectron tandem mass spectrometer

Glycated peptides arising from in vivo digestion of glycated proteins, usually called advanced glycation end (AGE) product peptides, are biologically relevant compounds due to their reactivity towards circulating and tissue proteins. To investigate their structures, in vitro glycation of human serum albumin (HSA) has been performed and followed by enzymatic digestion. Using different MALDI based approaches the digestion products obtained have been compared with those arising from enzymatic digestion of the protein. Results obtained using 2,5-dihydroxybenzoic acid (DHB) indicate this as the most effective matrix, leading to an increase in the coverage of the glycated protein. Off-line microbore liquid chromatography prior to MALDI analysis reveals that 63% of the free amino groups amenable to glycation are modified. In addition, the same approach shows the co-presence of underivatised peptides. This indicates that, regardless of the high glucose concentration employed for HSA incubation, glycation does not go to completion. Tandem mass spectrometric data suggest that the collision induced dissociation of singly charged glycated peptides leads to specific fragmentation pathways related to the condensed glucose molecule. The specific neutral losses derived from the activated glycated peptides can be used as signature for establishing the occurrence of glycation processes.     

An effective and rapid determination by MALDI/TOF/TOF of methionine sulphoxide content of ApoA‐I in type 2 diabetic patients

Increased oxidation of low density lipoprotein (LDL) is characteristic of atherosclerosis. In this frame, high density lipoproteins (HDL) play an important role, being able to remove lipid peroxides (LPOs) and cholesterol from oxidized LDL, so exhibiting a protective role against atherosclerosis. A wide range of reactive compounds lead to the oxidation of methionine (Met) residues with the formation of methionine sulphoxide (MetO) in apolipoprotein A‐I (ApoA‐I). Consequently, the determination of MetO level can give both an evaluation of oxidative stress and the reduced capability of ApoA‐I in LPOs and cholesterol transport. For these reasons, the development of analytical methods able to determine the MetO level is surely of interest, and we report here the results obtained by MALDI mass spectrometry. Copyright © 2013 John Wiley & Sons, Ltd.     

HPLC-electrospray tandem mass spectrometry for simultaneous quantitation of eight plasma aminothiols: Application to studies of diabetic nephropathy

It was reported that Hcy was related to the development of kidney disease, but it remains unknown whether Hcy is an independent biomarker for diabetic nephropathy. Analytical method for simultaneous determination of aminothiols among the Hcy metabolic cycle is desirable to discover other potential biomarkers. A high-performance liquid chromatography-electrospray tandem mass spectrometric (HPLC-ESI-MS/MS) method was established for simultaneous quantitation of Cysteine (Cys), total homocysteine (tHcy), S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), cystathionine (Cysta), methionine (Met), glutathione (GSH) and cysteinylglycine (Cys-gly) in plasma with N-(2-mercaptopropionyl)-glycine (MPG) as internal standard. The method had simple pretreatment without derivatization and the chromatograms show better separation of the eight aminothiols and the analytic time was 20 min. The results demonstrated that it provided an excellent linearity for all analytes over their respective concentration ranges and illustrated excellent precision and plasma recovery as well. Then, the method was applied in the case-control study of patients with diabetes mellitus (DM) and diabetic nephropathy (DN). In conclusion, it is an effective method to quantitate the concentrations of aminothiols in the human plasma. SAH and SAM were suggested as better potential biomarkers of DM and DN.     

Mass spectrometric characterization of the zein protein composition in maize flour extracts upon protein separation by SDS‐PAGE and 2D gel electrophoresis

Highly homogenous α zein protein was isolated from maize kernels in an environment‐friendly process using 95% ethanol as solvent. Due to the polyploidy and genetic polymorphism of the plant source, the application of high resolution separation methods in conjunction with precise analytical methods, such as MALDI‐TOF‐MS, is required to accurately estimate homogeneity of products that contain natural zein protein. The α zein protein product revealed two main bands in SDS‐PAGE analysis, one at 25 kDa and other at 20 kDa apparent molecular mass. Yet, high resolution 2DE revealed approximately five protein spot groups in each row, the first at ca. 25 kDa and the second at ca. 20 kDa. Peptide mass fingerprinting data of the proteins in the two dominant SDS‐PAGE bands matched to 30 amino acid sequence entries out of 102 non‐redundant data base entries. MALDI‐TOF‐MS peptide mapping of the proteins from all spots indicated the presence of only α zein proteins. The most prominent ion signals in the MALDI mass spectra of the protein mixture of the 25 kDa SDS gel band after in‐gel digestion were found at m/z 1272.6 and m/z 2009.1, and the most prominent ion signals of the protein mixture of the 20 kDa band after in‐gel digestion were recorded at m/z 1083.5 and m/z 1691.8. These ion signals have been found typical for α zein proteins and may serve as marker ion signals which upon chymotryptic digestion reliably indicate the presence of α zein protein in two hybrid corn products.     

Multivariate analysis approach to the plasma protein profile of patients with advanced colorectal cancer

The aim of the present study was to identify the pattern of plasma protein species of interest as markers of colorectal cancer (CRC). Using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS), the plasma protein profile was determined in nine stage IV CRC patients (study group) and nine clean-colon healthy subjects (control group). Multivariate analysis methods were employed to identify distinctive disease patterns at protein spectrum. In the study and control groups, cluster analysis (CA) on the complete MALDI-MS spectra plasma protein profile showed a distinction between CRC patients and healthy subjects, thus allowing the identification of the most discriminating ionic species. Principal component analysis (PCA) and linear discriminant analysis (LDA) yielded similar grouping results. LDA with leave-one-out cross validation achieved a correct classification rate of 89% in both the patients and the healthy subjects.     

Quantitative determination of metformin,glyburide and its metabolites in plasma and urine of pregnant patients by LC‐MS/MS

This report describes the development and validation of an LC‐MS/MS method for the quantitative determination of glyburide (GLB), its five metabolites (M1, M2a, M2b, M3 and M4) and metformin (MET) in plasma and urine of pregnant patients under treatment with a combination of the two medications. The extraction recovery of the analytes from plasma samples was 87–99%, and that from urine samples was 85–95%. The differences in retention times among the analytes and the wide range of the concentrations of the medications and their metabolites in plasma and urine patient samples required the development of three LC methods. The lower limit of quantitation (LLOQ) of the analytes in plasma samples was as follows: GLB, 1.02 ng/mL; its five metabolites, 0.100–0.113 ng/mL; and MET, 4.95 ng/mL. The LLOQ in urine samples was 0.0594 ng/mL for GLB, 0.984–1.02 ng/mL for its five metabolites and 30.0 µg/mL for MET. The relative deviation of this method was <14% for intra‐day and inter‐day assays in plasma and urine samples, and the accuracy was 86–114% in plasma, and 94–105% in urine. The method described in this report was successfully utilized for determining the concentrations of the two medications in patient plasma and urine. Copyright © 2014 John Wiley & Sons, Ltd.     

Use of MDLC-DIGE and LC-MS/MS to identify serum biomarkers for complete remission in patients with acute myeloid leukemia

The response criteria for complete remission (CR) in acute myeloid leukemia (AML) are currently based on morphology and blood cell counts. However, these criteria are insufficient to establish a diagnosis in cases with poor quality bone marrow (BM) samples demonstrating a loss of cellular morphology. We investigated whether the sera of patients contained biomarkers that indicate disease response status. First, we performed multidimensional liquid chromatography-differential gel electrophoresis (MDLC-DIGE) to generate protein profiles of two pooled, paired serum samples from patients who had achieved CR; one collected at diagnosis (PreCR) and the other collected after chemotherapy (CR). Then, with the biomarker candidates found, ELISA was carried out for individual PreCR and CR samples, and for other verification sets including nonremission (NR) patients and normal samples. We selected two proteins, complement factor H (CFH) and apolipoprotein H (ApoH), with dye (Cy) ratios showing greater than 2.0-fold differences between the pooled samples. ELISA showed that CFH and ApoH are useful for distinguishing between the recovered (CR and normal) and nonrecovered (PreCR, PreNR, and NR) states in AML (p <0.001). We successfully applied a protein profiling technology of MDLC-DIGE and LC-MS/MS to discover two biomarkers for CR which needs further validation for a clinical setting.     

Fast quantitative determination of methylphenidate levels in rat plasma and brain ex vivo by MALDI‐MS/MS

This study presents a simple and sensitive high‐throughput matrix‐assisted laser desorption/ionization time‐of‐flight tandem mass spectrometry (MALDI‐MS/MS) method for ex vivo quantification of methylphenidate (MPH) in rat plasma and brain. The common MALDI matrix alpha‐cyano‐4‐hydroxycinnamic acid was used to obtain an optimal dried droplet preparation. For method validation, standards diluted in plasma and brain homogenate prepared from untreated (control) rats were used. MPH was quantified within a concentration range of 0.1–40 ng/ml in plasma and 0.4–40 ng/ml in brain homogenate with an excellent linearity (R² ≥ 0.9997) and good precision. The intra‐day and inter‐day accuracies fulfilled the FDA's ±15/20 critera. The recovery of MPH ranged from 93.8 to 98.5% and 87.2 to 99.8% in plasma and homogenate, respectively. We show that MPH is successfully quantified in plasma and brain homogenate of rats pre‐treated with this drug using the internal standard calibration method. By means of this method, a linear correlation between plasma and brain concentration of MPH in rodents pre‐treated with MPH was detected. The simple sample preparation based on liquid‐liquid extraction and MALDI‐MS/MS measurement requires approximately 10 s per sample, and this significantly reduces analysis time compared with other analytical methods. To the best of our knowledge, this is the first MALDI‐MS/MS method for quantification of MPH in rat plasma and brain. Copyright © 2015 John Wiley & Sons, Ltd.     

Mass spectrometric characterization of low-molecular-mass color pI markers and their use for direct determination of pI value of proteins

The use of low-molecular-mass color pI markers for the determination of pI values of proteins in gel isoelectric focusing (IEF) in combination with mass spectrometry is described. Different types of substituted phenols of known pI values within the mass range 250-400 were used here as pI markers. The pure, synthesized pI markers were studied by MALDI-TOF/TOF MS. Fragmentation studies of the pI markers were also performed. Only stable and well-characterized pI markers were used in this work. The selected pI markers were mixed with proteins, deposited on a gel and separated in a pH gradient. Color pI markers enable supervision of progress of the focusing process and also estimation of the position of the invisible focused bands. The separated bands of the pI markers (containing separated proteins) were excised, and the pI markers were eluted from each gel piece by water/ethanol and identified by MALDI-TOF/TOF MS. From the washed gel pieces the remaining carrier ampholytes were then washed out and proteins were in-gel digested with trypsin. The obtained peptides were measured by MALDI-TOF/TOF MS and the proteins identified via a protein database search. This procedure allows avoiding time-consuming protein staining and destaining procedures, which shortens the analysis time roughly by half. For comparison, IEF gels were stained with Coomassie Brilliant Blue R 250 and proteins in the gel bands were identified according to the standard proteomic protocol. This work has confirmed that our approach can give information about the correct pI values of particular proteins and shorten significantly the time of analysis.     

胶内差异双向电泳-电喷雾串联质谱筛选UVB损伤皮肤细胞潜在的蛋白质靶标

联用胶内差异双向电泳(2D-DIGE)和高效液相色谱-电喷雾串联质谱(HPLC-nESI MS/MS)鉴定人角质形成细胞HaCaT应答中波紫外线(UVB)损伤的差异表达蛋白,筛选UVB影响皮肤细胞正常生理功能潜在的靶标蛋白.结果表明:UVB辐射明显影响HaCaT细胞的蛋白质表达谱,DeCyder软件在每块DIGE凝胶上...     

LC‐MS/MS determination of bakkenolide D in rats plasma and its application in pharmacokinetic studies

A sensitive and rapid liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method was developed and validated for determination of bakkenolide D (BD), which was further applied to assess the pharmacokinetics of BD. In the LC‐MS/MS method, the multiple reaction monitoring mode was used and columbianadin was chosen as internal standard. The method was validated over the range of 1–800 ng/mL with a determination coefficient >0.999. The lower limit of quantification was 1 ng/mL in plasma. The intra‐ and inter‐day accuracies for BD were 91–113 and 100–104%, respectively, and the inter‐day precision was <15%. After a single oral dose of 10 mg/kg of BD, the mean peak plasma concentration of BD was 10.1 ± 9.8 ng/mL at 2 h. The area under the plasma concentration–time curve (AUC_{0–24 h}) was 72.1 ± 8.59 h ng/mL, and the elimination half‐life (T_1/2) was 11.8 ± 1.9 h. In case of intravenous administration of BD at a dosage of 1 mg/kg, the AUC_{0–24 h} was 281 ± 98.4 h?ng/mL, and the T_1/2 was 8.79 ± 0.63 h. Based on these results, the oral bioavailability of BD in rats at 10 mg/kg is 2.57%. Copyright © 2013 John Wiley & Sons, Ltd.     

A rapid and sensitive LC‐MS/MS method for the determination of Pulsatilla saponin D in rat plasma and its application in a rat pharmacokinetic and bioavailability study

A simple, sensitive and specific liquid chromatography–tandem mass spectrometry method was developed and validated for the determination of Pulsatilla saponin D, a potential antitumor constituent isolated from Pulsatilla chinensis in rat plasma. Rat plasma samples were pretreated by protein precipitation with methanol. The method validation was performed in accordance with US Food and Drug Administration guidelines and the results met the acceptance criteria. The method was successfully applied to assess the pharmacokinetics and oral bioavailability of Pulsatilla saponin D in rats. Copyright © 2014 John Wiley & Sons, Ltd.     

高效液相色谱法测定糖尿病肾病患者血液中的非蛋白氮

采用反相高效液相色谱法对糖尿病患者血液中与肾病相关的四种非蛋白氮代谢产物（肌酸、肌酐、尿素和尿酸）进行了测定。获得的优化条件为：采用C18反相色谱柱,在室温下,以含10mmol／L,pH6．86的KH2PO4和体积分数30％的甲醇水溶液为流动相,流速0．9mL／min,检测波长200nm。在优化的条件下。5min内可对病人血液中上述4种物质同时进行测定。方法具有较好的重现性（迁移时间和峰面积的RSD均为3％）和较高的灵敏度以及较宽的线性范围（肌酸：5—300μmol／L,肌酐：10—200μmol／L,尿素：1～30mmol／L,尿酸：10—1500μmol／L）,适用于临床上病人血液的日常分析和早期肾病的监测。     

High MS-compatibility of silver nitrate-stained protein spots from 2-DE gels using ZipPlates and AnchorChips for successful protein identification

The availability of easy-to-handle, sensitive, and cost-effective protein staining protocols for 2-DE, in conjunction with a high compatibility for subsequent MS analysis, is still a prerequisite for successful proteome research. In this article we describe a quick and easy-to-use methodological protocol based on sensitive, homogeneous, and MS-compatible silver nitrate protein staining, in combination with an in-gel digestion, employing the Millipore 96-well ZipPlate system for peptide preparation. The improved quality and MS compatibility of the generated protein digests, as compared to the otherwise weakly MS-compatible silver nitrate staining, were evaluated on real tissue samples by analyzing 192 Coomassie-stained protein spots against their counterparts from a silver-stained 2-DE gel. Furthermore, the applicability of the experimental setup was evaluated and demonstrated by the analysis of a large-scale MALDI-TOF MS experiment, in which we analyzed an additional ~1000 protein spots from 2-DE gels from mouse liver and mouse brain tissue.     

Quantification of heteroclitin D in rat plasma: validation of an LC/MS/MS method and its application in a preclinical pharmacokinetic study

A rapid, sensitive and specific liquid chromatography tandem mass spectrometry (LC/MS/MS) method was developed and validated for the quantification of heteroclitin D in rat plasma after using gambogic acid as internal standard (IS). Chromatographic separation was done on a Thermo Hypersil GOLD column (30 × 2.1 mm, 3 µm) using a mobile phase consisting of methanol–water–formic acid (80:20:0.1, v/v/v). The mass spectrometer worked with positive electrospray ionization in multiple reaction monitoring mode, using target ions at [M + H]⁺ m/z 483.3 for heteroclitin D and [M + H]⁺ m/z 629.3 for the IS. The standard curve was linear (R² ≥0.995) over the concentration range 9.98–2080 ng/mL and had good back‐calculated accuracy and precision. The intra‐ and interday precision and accuracy determined on three quality control samples (29.94, 166.4 and 1872 ng/mL) were ≤12.8 and –8.9–3.6%, respectively. The extraction recovery was ≥88.2% and the lower limit of quantification was 9.98 ng/mL. The method was successfully applied to evaluate pharmacokinetics of heteroclitin D in Sprague–Dawley rats following a single intravenous bolus injection of 2.0 mg/kg heteroclitin. Copyright © 2014 John Wiley & Sons, Ltd.     

2D-DIGE-HPLC-nESIMS/MS对2,4-二硝基苯磺酸损伤HaCaT细胞差异蛋白质的鉴定

采用Cy2、Cy3和Cy5荧光染料标记蛋白,建立了人角质形成细胞HaCaT受2,4-二硝基苯磺酸(DNBS)刺激前后的双向胶内差异凝胶电泳(2D-DIGE)图谱,每组平行样本数为3。凝胶采用蛋白荧光染料Deep Purple进行后染色(Post-stain)。DeCyder定量分析软件在每块凝胶上平均检测到1 200个以上蛋白斑点,每块胶上都匹配得到的相同蛋白质斑点有846个。其中有7个斑点丰度变化在50%以上,统计学意义显著(P值小于0.05)。利用高效液相色谱-电喷雾串联质谱(HPLC-nESI MS/MS)成功鉴定5个表达上调的斑点分别为X染色体开放阅读框26(Cxorf26)、人辅分子伴侣23(PTGES3)、钙调蛋白(CALM3)、肌球蛋白轻链6(MYL6)和断裂点丛集区蛋白1(BANF1);2个表达下调蛋白斑点被鉴定为转录延伸因子B肽链2(TCEB2)和核糖体蛋白L23(RPL23)。除MYL6被报道与皮肤疾病相关外,其它蛋白与皮肤病变的关系有待研究。该研究得到的7个差异表达蛋白为DNBS类化学致癌物职业接触者皮肤病变研究提供了有价值的线索。     

Purification and characterization of a native zinc-binding high molecular weight multiprotein complex from human seminal plasma

The seminal plasma comprises secretions from various accessory sex glands. During fertilization spermatozoa undergo complex sequences of precisely timed events that are regulated by the activation of different intracellular signaling pathways. The precision and efficacy of these pathways are often influenced by the assembly and interactions of multiprotein complexes, thereby directing the flow of regulatory information. Our knowledge about these protein complexes present in human seminal plasma (HuSP) is limited. Here we report the identification and characterization of a native high molecular weight zinc-binding multiprotein complex from HuSP by utilizing 2-DE followed by MS. Twenty-six proteins representing isoforms and/or fragments of 11 different proteins were found to be assembled in this complex. Prostate-specific antigen, zinc α2-glycoprotein, prostatic acid phosphatase, and prolactin inducible protein were the major proteins of this complex. Dynamic light scattering experiments revealed changes in aggregation pattern accompanied with deviation from physiological pH and in presence of SDS. However, no significant changes were observed in the presence of physiological ligands such as zinc and fructose. The present study will be useful and contribute to guide the future studies performed for elucidation of biological significance of this native complex in HuSP.     

Comparative evaluation of five protocols for protein extraction from stony corals (Scleractinia) for proteomics

Corals especially the reef‐building species are very important to marine ecosystems. Proteomics has been used for researches on coral diseases, bleaching and responses to the environment change. A robust and versatile protein extraction protocol is required for coral proteomics. However, a comparative evaluation of different protein extraction protocols is still not available for proteomic analysis of stony corals. In the present study, five protocols were compared for protein extraction from stony corals. The five protocols were TRIzol, phenol‐based extraction (PBE), trichloroacetic acid (TCA)‐acetone, glass bead‐assisted extraction (GBAE) and a commercially available kit. PBE, TRIzol and the commercial kit were more robust for extracting proteins from stony corals. The protein extraction efficiency and repeatability, two dimensional electrophoresis (2‐DE) and matrix‐assisted laser desorption/ionization time of flight mass spectrometry (MALDI TOF MS) were employed to evaluate the protocols. The results indicated that PBE protocol had the better protein extraction efficiency than the others. Protein extraction coverage varied among the procedures. Each protocol favored for certain proteins. Therefore, it is very important for coral proteomic analysis to select a suitable protein protocol upon the experimental design. In general, PBE protocol can be the first choice for extracting proteins from stony corals.