Proteomic analysis of the human colon carcinoma cell line (LIM 1215): development of a membrane protein database

The proteomic definition of plasma membrane proteins is an important initial step in searching for novel tumor marker proteins expressed during the different stages of cancer progression. However, due to the charge heterogeneity and poor solubility of membrane-associated proteins this subsection of the cell's proteome is often refractory to two-dimensional electrophoresis (2-DE), the current paradigm technology for studying protein expression profiles. Here, we describe a non-2-DE method for identifying membrane proteins. Proteins from an enriched membrane preparation of the human colorectal carcinoma cell line LIM1215 were initially fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 4-20%). The unstained gel was cut into 16 x 3 mm slices, and peptide mixtures resulting from in-gel tryptic digestion of each slice were individually subjected to capillary-column reversed phase-high performance liquid chromatography (RP-HPLC) coupled with electrospray ionization-ion trap-mass spectrometry (ESI-IT-MS). Interrogation of genomic databases with the resulting collision-induced dissociation (CID) generated peptide ion fragment data was used to identify the proteins in each gel slice. Over 284 proteins (including 92 membrane proteins) were identified, including many integral membrane proteins not previously identified by 2-DE, many proteins seen at the genomic level only, as well as several proteins identified by expressed sequence tags (ESTs) only. Additionally, a number of peptides, identified by de novo MS sequence analysis, have not been described in the databases. Further, a "targeted" ion approach was used to unambiguously identify known low-abundance plasma membrane proteins, using the membrane-associated A33 antigen, a gastrointestinal-specific epithelial cell protein, as an example. Following localization of the A33 antigen in the gel by immunoblotting, ions corresponding to the theoretical A33 antigen tryptic peptide masses were selected using an "inclusion" mass list for automated sequence analysis. Six peptides corresponding to the A33 antigen, present at levels well below those accessible using the standard automated "nontargeted" approach, were identified. The membrane protein database may be accessed via the World Wide Web (WWW) at http://www.ludwig. edu.au/jpsl/jpslhome.html.     

Determination of minor proteins of bovine milk and colostrum by optical biosensor analysis

Automated, rapid, sensitive, and label-free biosensor-based immunoassays for immunoglobulin G (IgG), folate binding protein, lactoferrin, and lactoperoxidase in bovine milk using surface plasmon resonance optical detection with direct binding assay format are described. Samples are prepared for analysis by direct dilution into buffer. Analysis conditions, including ligand immobilization, flow rate, contact time, and regeneration are defined and nonspecific binding considerations evaluated. The technique has been applied to the measurement of these proteins in consumer milks, colostrum, milk products, and infant formulas, and their temporal change during early bovine lactation followed.     

Variations in trace element concentrations in breast milk with stages of lactation

The influence of stages of lactation on the mineral and trace elementconcentrations of breast milk collected from 20 healthy lactating women ina Nigeria population was investigated using instrumental neutron activationanalysis (INAA) and proton induced X-ray emission (PIXE) analysis. The breastmilk samples were divided into colostrum (day 1–5), transitional (day6–13) and mature milk (day 14–28). The result of the study showthat colostrum milk has a significantly higher mean concentration of Ca, Cl,Cu, Fe, Mg, Na and Zn than the transitional and mature milk. Furthermore,the mean concentrations of Fe, Mg, Na and Zn in transitional milk were foundto be significantly higher when compared with the mature milk. It was alsoobserved from the semi-log plot of the concentration of the elements againstdays of lactation that the concentration of all the elements decreased significantlythroughout the lactation period studied with Cu, Fe and Zn concentration showingthe greatest decline, particularly in the early stages of lactation.     

Application of capillary zone electrophoresis to the characterisation of the human milk protein profile and its evolution throughout lactation

This work describes the use of capillary zone electrophoresis for the characterisation of human milk proteins. The major proteins were identified following different strategies, such as the treatment with enzymes for selective protein modification. Using this method we studied the proteins in human milk from different donors throughout lactation. Qualitative and quantitative differences in the composition of the individual proteins were observed. The different beta-casein phosphoforms were separated and quantified. The average proportion of the 0P:1P:2P:3P:4P:5P was, approximately, 3:6:9:4:10:2. The evolution of the ratio of the different beta-casein phosphoforms during lactation is reported.     

Impact of prefractionation using Gradiflow on two-dimensional gel electrophoresis and protein identification by matrix assisted laser desorption/ionization-time of flight-mass spectrometry

Prefractionation of protein samples prior to two-dimensional electrophoresis (2-DE) has the potential to increase the dynamic detection range for proteomic analysis. We evaluated a membrane-based electrophoretic separation technique (Gradiflow) for its ability to fractionate an exoproteome sample from the filamentous fungus Trichoderma reesei. The sample was separated on the basis of size and charge. Buffer optimization was found to be necessary for successful size fractionation. Fractionation by charge was used to resolve the sample into four fractions that were subjected to analysis by two-dimensional electrophoresis (2-DE). Enhanced detection of low-abundance proteins with selective removal of high-abundance species was achieved. Fractionated and unfractionated samples were examined for differences in the ability to identify proteins following 2-DE using trypsin in-gel digestion followed by peptide mass fingerprinting using matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Fractionated samples showed marked improvement in protein identification ability and sequence coverage. This study demonstrates the utility of the Gradiflow for fractionation, resulting in an enhancement of resolution and characterization of a moderately complex proteome.     

Prefractionation of protein samples for proteome analysis using reversed-phase high-performance liquid chromatography

We describe an approach for fractionating complex protein samples prior to two-dimensional gel electrophoresis using reversed-phase high-performance liquid chromatography. Whole lysates of cells and tissue were prefractionated by reversed-phase chromatography and elution with a five-step gradient of increasing acetonitrile concentrations. The proteins obtained at each step were subsequently separated by high-resolution two-dimensional gel electrophoresis (2-DE). The reproducibility of this prefractionation technique proved to be optimal for comparing 2-DE gels from two different cell states. In addition, this method is suitable for enriching low-abundance proteins barely detectable by silver staining to amounts that can be detected by Coomassie blue and further analyzed by mass spectrometry.     

Towards a two-dimensional database of common human proteins.

A protein pattern of common human proteins was constructed by comparing the two-dimensional gel electrophoresis (2-DE) protein patterns from five cell lines of different germ layers. Total cell lysate and the isolated and purified nuclei of each cell line were separated by parallel electrophoresis runs in a multiple casting system of highest reproducibility. The computerized image analysis of the digitized 2-DE gels revealed a master protein pattern for each cell line. By comparison of all master protein patterns a 2-DE protein map of only common human proteins was constructed as a basis for a new 2-DE database. In a first step we have started characterizing a number of spots by microsequencing, amino acid composition analysis, and mass spectroscopy.     

Determination of immunoglobulin G in bovine colostrum and milk by direct biosensor SPR-immunoassay

An automated biosensor surface-plasmon resonance-based assay was developed for the determination of immunoglobulin G (IgG) in bovine milk and colostrum with either goat or rabbit antibovine IgG or protein G used as detecting molecule. The method is configured as a direct and nonlabeled immunoassay, with quantitation against an authentic IgG calibrant. Whole colostrum or milk is prepared for analysis by dilution into buffer. Analysis conditions, including ligand immobilization, flowrate, contact time, and regeneration, were optimized, and nonspecific binding was evaluated. Performance parameters included working range of 15-10 000 ng/mL, method detection limit of 0.08 mg/mL, overall instrument response reproducibility relative standard deviation (RSD(R)) of 0.47%, mean between-run RSD(R) of 10.5% for colostrum, and surface stability over 200 analyses. The proposed method was compared with independent alternative methods. The technique was applied to the measurement of IgG content during early lactation transition from colostrum to milk, as well as in consumer milk, colostrum, and hyperimmune milk powders.     

液相等电聚焦结合双向凝胶电泳分离碱性蛋白

在蛋白组学研究中, 经典的双向凝胶电泳法(2-DE)对碱性蛋白及低丰度蛋白的分离存在技术障碍, 但预分离技术的应用可弥补其缺陷. 液相等电聚焦可有效地分离富集复杂蛋白样品. 碱性胶条用于2-DE可极大地提高蛋白上样量和凝胶分辨率. 将上述两种技术相结合用于碱性蛋白质和低丰度蛋白质的分离鉴定, 可使碱端区域双向凝胶图谱质量显著提高, 蛋白点更清晰且点数增多, 质谱鉴定确信度提高, 碱性蛋白和低丰度蛋白质谱鉴定成功率提高, 对于蛋白组学研究具有一定的意义.     

Peptic digestion of beta-casein. Time course and fate of possible bioactive peptides

Numerous peptides obtained by enzymatic digestion of food proteins have been reported to exhibit biological activities. In this study, the focus was placed on peptides of beta-casein from bovine milk after a gastro-analogous in vitro digestion with pepsin, a protease with broad specificity. In order to study the time course of the digestion, the process was stopped after specific times and the samples were subjected to HPLC separation followed by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) and nanoelectrospray (nanoESI) quadrupole time-of-flight (qTOF) mass spectrometry. A combined sequencing approach using de novo interpretation and databases was employed. Overall, 100% of the beta-casein sequence was covered by identifying 125 peptides of 4-84 residues in length, including 3 phosphorylated species. The results show that the peptic hydrolysis starts at the C-terminus of the protein. The release of known bioactive peptides from beta-casein following the peptic digestion under simulated gastric conditions is unlikely with a few exceptions. Furthermore, an amino acid variation was found, providing evidence for the existence of an additional genetic variant of beta-casein.     

A proteome database of human primary T helper cells

We have established the first public database of human primary T helper cell proteome using two-dimensional electrophoresis (2-DE) and matrix assisted laser desorption/ionization-time of flight-mass spectrometry. For the database, CD4+ human T cells were activated with anti-CD3+anti-CD28 antibodies and metabolically labeled with [35S]methionine for 24 h. Cells were lysed and proteins were separated by 2-DE. About 1500 protein spots were detected in the resulting 2-DE gels with silver staining, and 2000 spots with autoradiography. We have identified 91 proteins from the 2-DE gels using peptide mass fingerprinting, and annotated them to our database. The identified proteins are also linked to SWISS-PROTand NCBI protein databases. Our database is available via the Internet at http://www3.btk.utu.fi:8080/Genomics/Proteomics/Database.     

Detection and characterization by high-performance liquid chromatography and mass spectrometry of a goat beta-casein associated with a CSN2 null allele

The identification and characterization of a truncated goat beta-casein, associated with a null beta-casein allele (CSN2(O')), is reported. The truncated beta-casein predicted at the DNA level (NCBI Acc. No. CAB39313) but never observed at the protein level, here named beta-casein O, was detected as a minor component in a goat milk sample from an autochthonous breed from southern Italy, 'Rossa Mediterranea', by reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry (RP-HPLC/ESI-MS). The ESI mass spectrum of the intact beta-casein O determined an M(r) value of 18 780 Da (calculated 18 781.5). Characterization of the amino acid sequence, performed by coupling trypsin digestion with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), RP-HPLC/ESI-MS and tandem mass spectrometry (MS/MS), demonstrated that the amino acid sequence corresponds to the 1-166 sequence of mature beta-casein variant A (Acc. No. P33048), thus confirming that the protein is coded by the null allele CSN2(O'), characterized by a transition (C --> T) at the 373rd nucleotide of the 7th exon of the gene, which generates a premature stop codon in position 182.     

Human proteome enhancement: high-recovery method and improved two-dimensional map of colostral fat globule membrane proteins.

The human milk fat globule membrane protein composition is still largely unknown, although it counts for 2-4% of the total milk protein content and contains several important biologically active components. The aim of this work was to create a two-dimensional electrophoresis (2-DE) map of the human milk fat globule membrane proteins, both integral and membrane-associated, and to identify and characterize as many protein components as possible. A new protocol for the solubilization and extraction of the human milk fat globule membrane proteins with a double extraction procedure is presented, and the results compared with the extraction methods reported in the literature. The proteins were separated, in the first dimension, by isoelectric focusing (IEF) in the pH range 3-10 on strips of 13 cm length and, in the second dimension, by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 11.5% T homogeneous gels. A reproducible 2-DE map of integral and membrane-associated proteins was obtained and the first 23 spots, representing the major components, were identified by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometric analysis and/or by amino acid sequencing.     

Analysis of recombinant and modified proteins by capillary zone electrophoresis coupled with electrospray ionization tandem mass spectrometry.

A method for rapid characterization of recombinant and modified proteins with known sequences is described. The analytical system consists of a capillary zone electrophoresis (CZE) instrument coupled to an electrospray ionization ion trap tandem mass spectrometer via a sheath-flow interface. Following the procedure consists of proteolytic fragmentation, CZE peptide separation, tandem mass spectrometry (MS-MS) analysis of separated peptides, sequence database search and monitoring of the specific peptides, C 125 S mutated interleukin 2 (S-125-IL2) and bovine beta-casein were characterized as a model of recombinant protein and naturally modified protein, respectively. A tryptic peptide mixture derived from the synthetic salmon calcitonin (s-CT) was also analyzed to test the performance of the system. Although a conventional sheath-flow interface with much higher flow-rate compared to the microspray interface and nanospray interface was used, the proteins were identified at the low picomole level.     

Iterative data analysis is the key for exhaustive analysis of peptide mass fingerprints from proteins separated by two-dimensional electrophoresis

Peptide mass fingerprinting (PMF) is a powerful tool for identification of proteins separated by two-dimensional electrophoresis (2-DE). With the increase in sensitivity of peptide mass determination it becomes obvious that even spots looking well separated on a 2-DE gel may consist of several proteins. As a result the number of mass peaks in PMFs increased dramatically leaving many unassigned after a first database search. A number of these are caused by experiment-specific contaminants or by neighbor spots, as well as by additional proteins or post-translational modifications. To understand the complete protein composition of a spot we suggest an iterative procedure based on large numbers of PMFs, exemplified by PMFs of 480 Helicobacter pylori protein spots. Three key iterations were applied: (1) Elimination of contaminant mass peaks determined by MS-Screener (a software developed for this purpose) followed by reanalysis; (2) neighbor spot mass peak determination by cluster analysis, elimination from the peak list and repeated search; (3) re-evaluation of contaminant peaks. The quality of the identification was improved and spots previously unidentified were assigned to proteins. Eight additional spots were identified with this procedure, increasing the total number of identified spots to 455.     

Identification of a serine protease inhibitor homologue in Bird's Nest by an integrated proteomics approach.

For centuries, the edible nests of Collocalia spp. ("Bird's Nests") have been used as a Chinese delicacy that had been claimed to be an effective health-giving tonic. However, clinical studies indicated that in Singapore, Bird's Nest is the most common cause of food-induced anaphylaxis in children, which could lead to potentially life-threatening allergenic reactions. The purpose of this study was to characterize the major allergens in Bird's Nest by using the combined technologies of two-dimensional gel electrophoresis (2-DE), immunochemistry, N-terminal protein sequencing, and mass spectrometry. Results from the immunostaining of the Western blots of the Bird's Nest 2-DE separated proteins with the sera from allergic patients indicated the presence of a major allergen of 66 kDa. Initial searches of the matrix assisted laser desorption/ionization--time of flight--mass spectrometry (MALDI-TOF-MS) tryptic peptide masses of the allergen in the SWISS-PROT and NCBI nonredundant databases revealed that this protein was novel. Based on the partial protein sequence information obtained from N-terminal microsequencing and nanoelectrospray-tandem MS, the 66 kDa immunoreactive allergen was found to be homologous to ovoinhibitor, a Kazal-type serine protease inhibitor, which is one of the dominant allergens found in chicken egg white.     

Isolation and identification of O- and N-linked glycoproteins in milk from different mammalian species and their roles in biological pathways which support infant growth

Milk serves as the sole nutrition for newborns, as well as a medium for the transfer of immunological components from the mother to the baby. This study reveals different glycoprotein profiles obtained from human, bovine, and caprine milk and their potential roles in supporting infant growth. Proteins from these three milk samples are separated and analyzed using two-dimensional gel electrophoresis (2-DE). Glycosylated proteins from all samples are enriched by affinity chromatography using lectins from the seeds of Artocarpus integer before analysis using LC/MS-QTOF. The glycoproteome profiling demonstrates that glycosylated proteins are higher in caprine milk compared to other samples. Analysis using LC/MS-QTOF identified 42 O-glycosylated and 56 N-glycosylated proteins, respectively. Among those identified, human milk has 17 glycoproteins, which are both O- and N-glycosylated, whereas caprine and bovine have 10 and 1, respectively. Only glycoproteins from human milk have shown positive matching to important human biological pathways, such as vesicle-mediated transport, immune system and hemostasis pathways. Human milk remains unique for human babies with the presence of antibodies in the form of immunoglobulins that are lacking in ruminant milk proteomes.     

Identification and quantification of major bovine milk proteins by liquid chromatography

In the field of food quality, bovine milk products are of particular interest due to the social and economic importance of the dairy products market. However, the risk of fraudulent manipulation is high in this area, for instance, replacing milk powder by whey is very interesting from an economic point of view. Therefore, there is a need to have suitable analytical methods available for the determination of all milk components, which is currently not the case, especially for the main proteins. The detection of potential manipulations requires then a clear analytical characterisation of each type of bovine milk, what constitutes the goal of this work. The separation of the major milk proteinic components has been carried out by ion-pair reversed-phase HPLC with photodiode array detection, using a C4 column. The overall optimisation has been achieved using a statistical experimental design procedure. The identification of each protein was ascertained using retention times, peak area ratios and second derivative UV spectra. Quantification was based on calibration curves drawn using purified proteins. Major sources of uncertainty were identified and the full uncertainty budget was established. The procedure was initially developed using the skimmed milk powder certified reference material CRM 063R and then applied to various types of commercial milks as well as to raw milk. The method is able to separate and quantify the seven major proteins (K-casein, alphas2-casein, alphas1-casein, beta-casein, alpha-lactalbumin, beta-lactoglobulin B and beta-lactoglobulin A) in one run and also to provide precise determinations of the total protein concentration. These are important results towards the further development of a reference method for major proteins in milk. In addition, the use of a certified material reference is suggested in order to make comparisons of method performances possible.     

Enhancement of phosphoprotein analysis using a fluorescent affinity tag and mass spectrometry

A fluorescent affinity tag (FAT) was synthesized and was utilized to selectively modify phosphorylated serine and threonine residues via beta-elimination and Michael addition chemistries in a 'one-step' reaction. This labeling technique was used for covalent modification of both phosphoproteins and phosphopeptides, allowing identification of these molecular species by fluorescence imaging after solution- or gel-based separation methods. In addition to the strong fluorescence of the rhodamine tag, a commercially available antibody can be used to enrich low-abundance post-labeled phosphopeptides present in complex mixtures. Application of this methodology to phosphorylation-site mapping has been evaluated for a phosphoprotein standard, bovine beta-casein. Initial results demonstrated low femtomole detection limits after fluorescence image analysis of FAT-labeled proteins or peptides.     

The establishment of a human liver nuclei two-dimensional electrophoresis reference map

This short communication describes the establishment of a two-dimensional electrophoresis (2-DE) reference map of nuclear proteins isolated from human liver. The human liver nuclei 2-DE reference map contains 1497 spots. In an initial identification study using peptide mass fingerprinting as a means of protein identification we were able to identify 26 spots corresponding to 15 different proteins. The human liver nuclei 2-DE reference map is now included in the SWISS-2DPAGE database, which can be accessed through the ExPASy server (http://www.expasy.ch/ch2d/).