Lipoproteomics II: mapping of proteins in high-density lipoprotein using two-dimensional gel electrophoresis and mass spectrometry

High-density lipoprotein (HDL) is the most abundant lipoprotein particle in the plasma and a negative risk factor of atherosclerosis. By using a proteomic approach it is possible to obtain detailed information about its protein content and protein modifications that may give new information about the physiological roles of HDL. In this study the two subfractions; HDL(2) and HDL(3), were isolated by two-step discontinuous density-gradient ultracentrifugation and the proteins were separated with two-dimensional gel electrophoresis and identified with peptide mass fingerprinting, using matrix-assisted laser desorption/ionisation time of flight mass spectrometry. Identified proteins in HDL were: the dominating apo A-I as six isoforms, four of them with a glycosylation pattern and one of them with retained propeptide, apolipoprotein (apo) A-II, apo A-IV, apo C-I, apo C-II, apo C-III (two isoforms), apo E (five isoforms), the recently discovered apo M (two isoforms), serum amyloid A (two isoforms) and serum amyloid A-IV (six isoforms). Furthermore, alpha-1-antitrypsin was identified in HDL for the first time. Additionally, salivary alpha-amylase was identified as two isoforms in HDL(2), and apo L and a glycosylated apo A-II were identified in HDL(3). Besides confirming the presence of different apolipoproteins, this study indicates new patterns of glycosylated apo A-I and apo A-II. Furthermore, the study reveals new proteins in HDL; alpha-1-antitrypsin and salivary alpha-amylase. Further investigations about these proteins may give new insight into the functional role of HDL in coronary artery diseases.     

Proteome analysis of human hepatocellular carcinoma tissues by two-dimensional difference gel electrophoresis and mass spectrometry

Proteome analysis of human hepatocellular carcinoma tissues was conducted using two-dimensional difference gel electrophoresis coupled with mass spectrometry. Paired samples from the normal and tumor region of resected human liver were labeled with Cy3 and Cy5, respectively while the pooled standard sample was labeled with Cy2. After analysis by the DeCyder software, protein spots that exhibited at least a two-fold difference in intensity were excised for in-gel tryptic digestion and matrix-assisted laser desorption/ionization-time of flight mass spectrometry. A total of 6 and 42 proteins were successfully identified from the well- and poorly-differentiated samples, respectively. The majority of these proteins are related to detoxification/oxidative stress and metabolism. Three down-regulated metabolic enzymes, methionine adenosyltransferase, glycine N-methyltransferase, and betaine-homocysteine S-methyltransferase that are involved in the methylation cycle in the liver are of special interest. Their expression levels, especially, methionine adenosyltransferase, seemed to have a major influence on the level of S-adenosylmethionine (AdoMet), a vital intermediate metabolite required for the proper functioning of the liver. Recent work has shown that chronic deficiency in AdoMet in the liver results in spontaneous development of steatohepatitis and hepatocellular carcinoma, and hence the down-regulation of hepatic methionine adenosyltransferase in our hepatocellular carcinoma samples is in line with this observation. Moreover, when a comparison is made between the differentially expressed proteins from our human hepatocellular carcinoma samples and from the liver tissues of knockout mice deficient in methionine adenosyltransferase, there is a fairly good correlation between them.     

Lipoproteomics I: mapping of proteins in low-density lipoprotein using two-dimensional gel electrophoresis and mass spectrometry

The molecular mechanisms underlying the relationship between low-density lipoprotein (LDL) and the risk of atherosclerosis are not clear. Therefore, detailed information about the protein composition of LDL may contribute to reveal its role in atherogenesis and the mechanisms that lead to coronary disease in humans. Here, we sought to map the proteins in human LDL by a proteomic approach. LDL was isolated by two-step discontinuous density-gradient ultracentrifugation and the proteins were separated with two-dimensional gel electrophoresis and identified with peptide mass fingerprinting, using matrix assisted laser desorption/ionization-time of flight-mass spectrometry and with amino acid sequencing using electrospray ionization tandem mass spectrometry. These procedures identified apo B-100, apo C-II, apo C-III (three isoforms), apo E (four isoforms), apo A-I (two isoforms), apo A-IV, apo J and apo M (three isoforms not previously described). In addition, three proteins that have not previously been identified in LDL were found: serum amyloid A-IV (two isoforms), calgranulin A, and lysozyme C. The identities of apo M, calgranulin A, and lysozyme C were confirmed by sequence information obtained after collision-induced dissociation fragmentation of peptides characteristic for these proteins. Moreover, the presence of lysozyme C was further corroborated by demonstrating enriched hydrolytic activity in LDL against Micrococcus lysodeikticus. These results indicate that in addition to the dominating apo B-100, LDL contains a number of other apolipoproteins, many of which occur in different isoforms. The demonstration, for the first time, that LDL contains calgranulin A and lysozyme C raises the possibility that LDL proteins may play hitherto unknown role(s) in immune and inflammatory reactions of the arterial wall.     

A new method to improve sensitivity and resolution in matrix-assisted laser desorption/ionization time of flight mass spectrometry

High detection sensitivity and resolution are two critical parameters for recording good peptide mass fingerprints (PMF) of low abundance proteins. This paper reports a mass spectrometry (MS) sample preparation technique that could improve sensitivity and resolution. By coating the MS steel target with a thin layer of pentadecafluorooctamido propyltrimethoxysilane, which was both polar and nonpolar solvent repellent, the transferred sample droplets on its surface were significantly smaller. As a result, the analyte of the peptide mixture became more concentrated and homogeneous, which helped to improve the sensitivity. The advantages of a modified MS target were documented by mass spectra improvement of attomole level standard peptides and silver-stained proteins from polyacrylamide gels. The mass signal of angiotensin II at 100 attomole was difficult to record on the conventional support, whereas it was easily detected on the modified one. The PMF of cytochrome C was also better recorded on the modified support, in terms of both signal-to-noise ratio and the number of detected peptides. When silver-stained proteins from two-dimensional electrophoresis gels were analyzed, in most cases more satisfactory peptide mass spectra were obtained from the modified support. Searching protein databases with more mass data from the improved PMFs, several unknown proteins were successfully identified.     

Large-scale identification of proteins in human salivary proteome by liquid chromatography/mass spectrometry and two-dimensional gel electrophoresis-mass spectrometry

Human saliva contains a large number of proteins and peptides (salivary proteome) that help maintain homeostasis in the oral cavity. Global analysis of human salivary proteome is important for understanding oral health and disease pathogenesis. In this study, large-scale identification of salivary proteins was demonstrated by using shotgun proteomics and two-dimensinal gel electrophoresis-mass spectrometry (2-DE-MS). For the shotgun approach, whole saliva proteins were prefractionated according to molecular weight. The smallest fraction, presumably containing salivary peptides, was directly separated by capillary liquid chromatography (LC). However, the large protein fractions were digested into peptides for subsequent LC separation. Separated peptides were analyzed by on-line electrospray tandem mass spectrometry (MS/MS) using a quadrupole-time of flight mass spectrometer, and the obtained spectra were automatically processed to search human protein sequence database for protein identification. Additionally, 2-DE was used to map out the proteins in whole saliva. Protein spots 105 in number were excised and in-gel digested; and the resulting peptide fragments were measured by matrix-assisted laser desorption/ionization-mass spectrometry and sequenced by LC-MS/MS for protein identification. In total, we cataloged 309 proteins from human whole saliva by using these two proteomic approaches.     

Proteome analysis of human colon cancer by two-dimensional difference gel electrophoresis and mass spectrometry

Two-dimensional difference gel electrophoresis (2-D DIGE) coupled with mass spectrometry (MS) was used to investigate tumor-specific changes in the proteome of human colorectal cancers and adjacent normal mucosa. For each of six patients with different stages of colon cancer, Cy5-labeled proteins isolated from tumor tissue were combined with Cy3-labeled proteins isolated from neighboring normal mucosa and separated on the same 2-D gel along with a Cy2-labeled mixture of all 12 normal/tumor samples as an internal standard. Over 1500 protein spot-features were analyzed in each paired normal/tumor comparison, and using DIGE technology with the mixed-sample internal standard, statistically significant quantitative comparisons of each protein abundance change could be made across multiple samples simultaneously without interference due to gel-to-gel variation. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and tandem (TOF/TOF) MS provided sensitive and accurate mass spectral data for database interrogation, resulting in the identification of 52 unique proteins (including redundancies due to proteolysis and post-translationally modified isoforms) that were changing in abundance across the cohort. Without the benefit of the Cy2-labeled 12 sample mixture internal standard, 42 of these proteins would have been overlooked due to the large degree of variation inherent between normal and tumor samples.     

Proteome analysis of silk gland proteins from the silkworm, Bombyx mori

The silk gland of Bombyx mori is an organ specialized for the synthesis and secretion of silk proteins. We report here the resolution of silk gland proteins by 2-DE and the identification of many of those proteins. This was accomplished by dissecting the glands into several sections, with each exhibiting more than 400 protein spots by 2-DE, of which 100 spots were excised and characterized by in-gel digestion followed by PMF. Ninety-three proteins were tentatively identified. These were then categorized into groups involved in silk protein secretion, transport, lipid metabolism, defense, etc. Western blotting of a 2-DE gel using an antibody of the carotenoid binding protein confirmed the presence of this protein in the silk gland. Proteins including fibroin L-chain and P25 were found as multiple isoforms, some of which contained differential amounts of phosphate residues as analyzed by on-probe dephosphorylation. The current analysis contributes to our understanding of proteins expressed by the silk gland not only of the model lepidopteran B. mori, but also to proteins from other silk-producing insects such as Philosamia cynthia ricini.     

三种前处理在基质辅助激光解析电离飞行时间质谱鉴定假丝酵母菌属中的应用比较

目的 比较3种前处理方法在基质辅助激光解析电离飞行时间质谱(MALDI TOF MS)鉴定假丝酵母菌属中的结果可靠性。 方法 以ITS测序鉴定结果为金标准,对临床分离的66株假丝酵母分别采用传统直涂法、改良直涂法和甲酸-乙腈蛋白提取法进行前处理,MALDI TOF MS鉴定,比较3种方法的Biotyper Log值,分析质谱图的差异。 结果 传统直涂法、改良直涂法和甲酸-乙腈提取法对66株假丝酵母的属水平鉴定率分别为48.5%、50.0%和97.0%,Biotyper Log均值分别为1.628、1.674和2.010,其中甲酸-乙腈提取法对66株假丝酵母的种水平鉴定率为53.0%。甲酸-乙腈提取法得到的质谱图比另2种方法的质谱图离子峰更加密集,图像更复杂,鉴定结果可信度更高。 结论 甲酸-乙腈蛋白提取法对假丝酵母菌属的鉴定成功率和可靠性明显高于传统直涂法和改良直涂法,对临床假丝酵母菌病的准确诊断具有重要价值。     

Nanoflow liquid chromatography coupled to matrix-assisted laser desorption/ionization mass spectrometry: sample preparation, data analysis, and application to the analysis of complex peptide mixtures

We report the development of a robust interface for off-line coupling of nano liquid chromatography (LC) to matrix-assisted laser desorption/ionisation-mass spectrometry (MALDI-MS) and its application to the analysis of proteolytic digests of proteins, both isolated and in mixtures. The interface makes use of prestructured MALDI sample supports to concentrate the effluent to a small sample plate area and localize the MALDI sample to a predefined array, thereby enriching the analyte molecules and facilitating automated MALDI-MS analysis. Parameters that influence the preparation of MALDI samples from the LC effluent were evaluated with regard to detection sensitivity, spectra quality, and reproducibility of the method. A procedure for data processing is described. The presented nano LC MALDI-MS system allowed the detection of several peptides from a tryptic digest of bovine serum albumin, at analyzed amounts corresponding to one femtomole of the digested protein. For the identification of native proteins isolated from mouse brain by two-dimensional gel electrophoresis, nano LC MALDI-MS increased the number of detected peptides, thereby allowing identification of proteins that could not be identified by direct MALDI-MS analysis. The ability to identify proteins in complex mixtures was evaluated for the analysis of Escherichia coli 50S ribosomal subunit. Out of the 33 expected proteins, 30 were identified by MALDI tandem time of flight fragment ion fingerprinting.     

Characterisation of bacteria by matrix-assisted laser desorption/ionisation and electrospray mass spectrometry

Chemical analysis for the characterisation of micro-organisms is rapidly evolving, after the recent advent of new ionisation methods in mass spectrometry (MS): electrospray (ES) and matrix-assisted laser desorption/ionisation (MALDI). These methods allow quick characterisation of micro-organisms, either directly or after minimum sample preparation. This review provides a brief introduction to ES and MALDI MS and a discussion of micro-organism characterisation capabilities. Some attention is devoted to the analysis of mixtures of proteins, lipids and other compounds, to the combination of polymerase chain reaction technology and MS, and to the analysis of whole bacteria and their lysates. The review of results produced hitherto is concluded with an outlook on future developments.     

Liquid ultraviolet matrix-assisted laser desorption/ionization -- mass spectrometry for automated proteomic analysis

We have combined several key sample preparation steps for the use of a liquid matrix system to provide high analytical sensitivity in automated ultraviolet -- matrix-assisted laser desorption/ionisation -- mass spectrometry (UV-MALDI-MS). This new sample preparation protocol employs a matrix-mixture which is based on the glycerol matrix-mixture described by Sze et al. The low-femtomole sensitivity that is achievable with this new preparation protocol enables proteomic analysis of protein digests comparable to solid-state matrix systems. For automated data acquisition and analysis, the MALDI performance of this liquid matrix surpasses the conventional solid-state MALDI matrices. Besides the inherent general advantages of liquid samples for automated sample preparation and data acquisition the use of the presented liquid matrix significantly reduces the extent of unspecific ion signals in peptide mass fingerprints compared to typically used solid matrices, such as 2,5-dihydroxybenzoic acid (DHB) or alpha-cyano-hydroxycinnamic acid (CHCA). In particular, matrix and low-mass ion signals and ion signals resulting from cation adduct formation are dramatically reduced. Consequently, the confidence level of protein identification by peptide mass mapping of in-solution and in-gel digests is generally higher.     

Analysis of derivatised steroids by matrix-assisted laser desorption/ionisation and post-source decay mass spectrometry

Neutral steroids are difficult to analyse using desorption ionisation methods coupled with mass spectrometry (MS). However, steroids with an unhindered ketone group can readily be derivatised with the Girard P (GP) reagent to give GP hydrazones. Steroid GP hydrazones contain a quaternary nitrogen atom and are readily desorbed in the matrix-assisted laser desorption/ionisation (MALDI) process, giving an improvement in sensitivity of two orders of magnitude. Steroids without a ketone group, but with a 3beta-hydroxy-Delta5 function, can be readily converted to 3-oxo-Delta4 steroids and subsequently derivatised to GP hydrazones for MALDI analysis. In addition to giving strong [M]+ ions upon MALDI, steroid GP hydrazones give informative post-source decay (PSD) spectra. By using the accurate mass of the precursor-ion measured by MALDI-MS, in combination with the structural information encoded in its PSD spectrum, steroid structures can readily be determined.     

Towards the proteome of Burkholderia cenocepacia H111: setting up a 2-DE reference map

Polyphasic-taxonomic studies of the past decade have shown that the Burkholderia cepacia complex (Bcc) comprises at least nine species, which share a high degree of 16S rDNA (98-100%) sequence similarity but only moderate levels of DNA-DNA hybridization. Members of the Bcc are well known as opportunistic pathogens of plants, animals and humans but also as biocontrol and bioremediation agents. In this study intra-, surface-associated and extracellular proteins of B. cenocepacia H111, which was isolated from a cystic fibrosis patient, were examined by 2-DE coupled to MALDI-TOF MS. MS and MS/MS data were searched against a database comprising all currently available annotated proteins of genetically closely related strains. In total 642 proteins spots were successfully identified corresponding to 390 different protein species, which were classified into functional categories. The majority of these proteins could be linked to housekeeping functions in energy production, amino acid metabolism, protein folding, post-translational modification and turnover, and translation. Noteworthy is the fact that a significant number of truly secreted and membrane proteins were identified in the extracellular and surface-associated sub-proteomes. This indicates that the pre-fractionation protocol used in this study is a highly valuable strategy for unravelling the cellular location of the identified proteins.     

A combination of protein profiling and isotopomer analysis using matrix-assisted laser desorption/ionization-time of flight mass spectrometry reveals an active metabolism of the extracellular matrix of 3T3-L1 adipocytes

Differential gel electrophoresis followed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) is a commonly used protein profiling method. However, observed changes can be explained in multiple ways, one of which is by the protein turnover rate. In order to easily and rapidly obtain information on both the identity and turnover of individual proteins, we applied a combination of protein labeling with L-(ring-2,3,4,5,6 2H5) phenylalanine and MALDI-TOF MS. While the spectrum reveals the identity of a protein, mass isotopomer analysis provides information about the rate of protein labeling as a measure of synthesis or turnover. Using this approach on mature 3T3-L1 adipocytes, we were able to discriminate between rapidly and slowly metabolised proteins. In our isolate, proteins of the cytoskeleton appeared to be slowly metabolised, whereas components of the extracellular matrix, in particular collagen type I alpha 1 (COL1A1) and collagen type I alpha 2 (COL1A2) showed rapid accumulation of newly synthesized proteins. Both proteins appeared to be metabolised in the same ratio as they are present in collagen fibers, i.e. 2:1 (COL1A1: COL1A2). In addition, functionally related proteins were also readily labeled. Taken together, we have shown that a combination of stable isotope labeling and protein profiling by gel electrophoresis and MALDI-TOF analysis can simultaneously provide information on the identity and relative metabolic rate of proteins in eukaryotic cells in a simple, nonhazardous and rapid-throughput way.     

Navigated laser capture microdissection as an alternative to direct histological staining for proteomic analysis of brain samples

Proteomic analysis of the brain is complicated by the need to obtain cells from specific anatomical regions, or nuclei. Laser capture microdissection (LCM) is a technique that is precise enough to dissect single cells within a tissue section, and thus could be useful for isolating specific brain nuclei for analysis. However, we and others have previously demonstrated that histological staining protocols used to guide LCM have detrimental effects on protein separation by two-dimensional electrophoresis (2-DE). Here we describe a new LCM method called navigated LCM. This microdissection method uses fixed but unstained tissue as starting material and thus enables us to avoid artifacts induced by tissue staining. By comparing 2-DE results obtained from fixed, unstained LCM brain tissue samples to those obtained from manually dissected samples, we demonstrated that this microdissection process gave similar protein recovery rates and similar resolution of protein spots on 2-DE gels. Moreover, matrix-assisted laser desorption/ionization-time of flight mass spectrometry analysis of selected spots from gels derived from control and fixed, LCM samples revealed that the fixation-LCM process had no effect on protein identification. Navigated LCM of tissue sections is therefore a practical and powerful method for performing proteomic studies in specifically defined brain regions.     

Differential analysis of Bacillus anthracis after pX01 plasmid curing and comprehensive data on Bacillus anthracis infection in macrophages and glial cells

Bacillus anthracis is a gram-positive bacterial organism responsible for anthrax. This organism has two pathogenic plasmids: pX01 and pX02. The genetic function of pX01, which comprises about 198 kb, is not known, except for a region called the pathogenic island, which contains three genes-pag, lef, and cya-that code for three toxic proteins. A 2-D difference gel electrophoresis (2-D DIGE) system was used to verify the existence of proteins controlled by the pX01 plasmid, and protein regulation data were obtained using DeCyder software. A total of 1728 proteins were identified in the wild-type strain of this organism and 1684 in the pX01 plasmid. Twenty-seven of these proteins disappeared and eight appeared when the pX01 plasmid was removed. An additional 52 proteins were downregulated and 15 were upregulated when this plasmid was removed. A total of 102 proteins have been identified using the MALDI-TOF method of analysis, including 49 whose functions are unknown. Among these, 31 participate in metabolic processes, two in cellular processes, 15 in the processing of genetic information, and five in the processing of extracellular information. Another seven proteins participate in bacterial virulence and pathogenesis. We investigated the functions of these proteins in other bacteria, particularly the B. anthracis derivative H9041. Bacterial growth differed between pX01+/pX02+ B. anthracis and its pX01-/pX02+ derivative as did the cytotoxicity of macrophages infected by pX01+/pX02+ B. anthracis and the pX01-pX02+ derivative. We also found that S100B protein levels increased in the host infected with pX01+/pX02+ B. anthracis or its pX01-/pX02+ derivative. These data suggest that the pX01 plasmid plays a key role in the regulation of protein functions in B. anthracis.     

Proteomic analysis of progressive factors in uterine cervical cancer

Human papillomavirus (HPV) infections play a crucial role in the progress of cervical cancer. The high-risk HPV types are frequently associated with the development of malignant lesions. Some of the latest studies have demonstrated that the high-risk HPV 16 and 18 are predominantly detected in the more aggressive cancers. In the present study, we aimed to establish the proteomic profiles and characterization of the tumor related proteins by using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). For proteomic analysis, patients infected by HPV 16 or 18 were included in this study. We compared nuclear protein and cytoplasmic protein, separately by using the subcellular fraction. Differential protein spots between cervical cancer with high-risk HPV, HPV 16 or HPV 18, and HaCaT cell lines were characterized by 2-DE. Those proteins analyzed by peptide mass fingerprinting based on MALDI-TOF MS and database searching were the products of oncogenes or proto-oncogenes, and the others were involved in the regulation of cell cycle, for general genomic stability, telomerase activation, and cell immortalization. However, there was no difference in protein characterization for cervical cancer between HPV 16 and HPV 18 infection. Nonetheless, these data are valuable for the mass identification of differentially expressed proteins involved in human uterine cervical cancer. Moreover, the data has enormous value for establishing the human uterine cervical cancer proteome database that can be used in screening a molecular marker for the further study of human uterine cervical cancer, and also for studying any correlation among the cancers induced by HPV.