Proteome analysis of Corynebacterium glutamicum.

By the use of different Corynebacterium glutamicum strains more than 1.4 million tons of amino acids, mainly L-glutamate and L-lysine, are produced per year. A project was started recently to elucidate the complete DNA sequence of this bacterium. In this communication we describe an approach to analyze the C. glutamicum proteome, based on this genetic information, by a combination of two-dimensional (2-D) gel electrophoresis and protein identification via microsequencing or mass spectrometry. We used these techniques to resolve proteins of C. glutamicum with the aim to establish 2-D protein maps as a tool for basic microbiology and for strain improvement. In order to analyze the C. glutamicum proteome, methods were established to fractionate the C. glutamicum proteins according to functional entities, i.e., cytoplasm, membranes, and cell wall. Protein spots of the cytoplasmic and membrane fraction were identified by N-terminal sequencing, immunodetection, matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and electrospray ionization-mass spectrometry (ESI-MS). Additionally, a protocol to analyze proteins secreted by C. glutamicum was established. Approximately 40 protein spots were observed on silver-stained 2-D gels, 12 of which were identified.     

Reliable automatic protein identification from matrix-assisted laser desorption/ionization mass spectrometric peptide fingerprints

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry of protein samples from two-dimensional (2-D) gels in conjunction with protein sequence database searches is frequently used to identify proteins. Moreover, the automatic analysis of complete 2-D gels with hundreds and even thousands of protein spots ("proteome analysis") is possible, without human intervention, with the availability of highly accurate mass spectrometry instruments, and high-throughput facilities for preparation and handling of protein samples from 2-D gels. However, the lack of software for precise automatic analysis and annotation of mass spectra, as well as software for in-batch sequence database queries, is increasingly becoming a significant bottleneck for the proteomics work flow. In the present paper we outline an algorithm for reliable, accurate, and automatic evaluation of mass spectrometric data and database searches. We show here that simply selecting from the sequence database the protein that has the most matching fragment masses often leads to false-positive results. Reliable protein identification is dependent on several parameters: the accuracy of fragment mass determination, the number of masses submitted for query, the mass distribution of query masses, the number of masses matching between sample and database protein, the size of the sequence database, and the kind and number of modifications considered. Using these parameters, we derive a simple statistical estimation that can be used to calculate the probability of true-positive protein identification.     

Enrichment of hydrophobic proteins via Triton X-114 phase partitioning and hydroxyapatite column chromatography for mass spectrometry

Membrane proteins are the starting point of several signal transduction pathways. Therefore, the separation and identification of these proteins are of great interest in proteome analysis. However, the specific properties of membrane proteins seriously impede their analysis. We present an effective and highly reproducible method for the two-dimensional separation of extremely hydrophobic proteins and demonstrate the advantages of special preseparation procedures for the identification of proteins which have very similar Mr and p/. Using the example of the integral membrane protein very low density lipoprotein (VLDL) receptor (NCBI Acc. # 1730111) and the soluble heat shock protein (HSP) 90 (NCBI Acc. # 386786) we present the applicability of a phase-separation system with Triton X-114. Using matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) of the protein spots after 2-D separation of the hydrophilic and the strongly hydrophobic protein fraction of human endothelial cells (ECV cell line), we were able to distinguish both proteins.     

A mass spectrometry approach for the study of deglycosylated proteins

Mass spectrometry (MS) analysis, after enzymatic or chemical deglycosylation, requires preparatory steps to remove salts and buffers. In this work, the glycosylated protein fetuin and a lectin protein isolated from the serum of Alligator mississippiensis were used to evaluate methods for desalting samples after an enzymatic or chemical deglycosylation. Precipitation and dialysis were used to prepare the deglycosylated samples for MS analysis. Both the precipitation and dialysis methods were suitable for sample preparation prior to analysis by matrix assisted laser desorption ionization (MALDI) MS.     

Functional characterization of two-dimensional gel-separated proteins using sequential staining

Proteins separated by two-dimensional (2-D) gel electrophoresis can be visualized using various protein staining methods. This is followed by downstream procedures, such as image analysis, gel spot cutting, protein digestion, and mass spectrometry (MS), to characterize protein expression profiles within cells, tissues, organisms, or body fluids. Characterizing specific post-translational modifications on proteins using MS of peptide fragments is difficult and labor-intensive. Recently, specific staining methods have been developed and merged into the 2-D gel platform so that not only general protein patterns but also patterns of phosphorylated and glycosylated proteins can be obtained. We used the new Pro-Q Diamond phosphoprotein dye technology for the fluorescent detection of phosphoproteins directly in 2-D gels of mouse leukocyte proteins, and Pro-Q Emerald 488 glycoprotein dye to detect glycoproteins. These two fluorescent stains are compatible with general protein stains, such as SYPRO Ruby stain. We devised a sequential procedure using Pro-Q Diamond (phosphoprotein), followed by Pro-Q Emerald 488 (glycoprotein), followed by SYPRO Ruby stain (general protein stain), and finally silver stain for total protein profile. This multiple staining of the proteins in a single gel provided parallel determination of protein expression and preliminary characterization of post-translational modifications of proteins in individual spots on 2-D gels. Although this method does not provide the same degree of certainty as traditional MS methods of characterizing post-translational modifications, it is much simpler, faster, and does not require sophisticated equipment and expertise in MS.     

Proteome and phosphoproteome of primary cultured pig urothelial cells

Epithelial tissue lining the inner side of the urinary bladder is the most common target for bladder cancer-related diseases. Bladders of freshly slaughtered pigs were utilised for a comprehensive analysis of the proteome and phosphoproteome of bladder epithelial cells. Following protein separation by 2-D gel electrophoresis and identification by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) the first proteome and phosphoproteome maps of pig urinary bladder epithelial cells (PUBEC) were established. A total of 120 selected protein spots were identified. By using the La(3+) enrichment method further developed in our laboratory we identified 31 phosphoproteins with minimal contamination by non-phosphopeptides. The 2-DE map of pig urothelial cells may prove as a useful tool for studies on uroepithelial biology, and the analysed phosphoproteins expression pattern, together with the whole cell proteome, will be helpful for identifying the proteins involved in bladder-related diseases.     

Identification of proteins in human cerebrospinal fluid using liquid-phase isoelectric focusing as a prefractionation step followed by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionisation mass spectrometry

Cerebrospinal fluid (CSF) is in close proximity to the brain and changes in the protein composition of CSF may be indicative of altered brain protein expression in neurodegenerative disorders. Analysis of brain-specific proteins in CSF is complicated by the fact that most CSF proteins are derived from the plasma and tend to obscure less abundant proteins. By adopting a prefractionation step prior to two-dimensional gel electrophoresis (2-DE), less abundant proteins are enriched and can be detected in complex proteomes such as CSF. We have developed a method in which liquid-phase isoelectric focusing (IEF) is used to prefractionate individual CSF samples; selected IEF fractions are then analysed on SYPRO-Ruby-stained 2-D gels, with final protein identification by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS). To optimise the focusing of the protein spots on the 2-D gel, the ampholyte concentration in liquid-phase IEF was minimised and the focusing time in the first dimension was increased. When comparing 2-D gels from individual prefractionated and unfractionated CSF samples it is evident that individual protein spots are larger and contain more protein after prefractionation of CSF. Generally, more protein spots were also detected in the 2-D gels from prefractionated CSF compared with direct 2-DE separations of CSF. Several proteins, including cystatin C, IgM-kappa, hemopexin, acetyl-coenzyme A carboxylase-alpha, and alpha-1-acid glycoprotein, were identified in prefractionated CSF but not in unfractionated CSF. Low abundant forms of posttranslationally modified proteins, e.g. alpha-1-acid glycoprotein and alpha-2-HS glycoprotein, can be enriched, thus better resolved and detected on the 2-D gel. Liquid-phase IEF, as a prefractionation step prior to 2-DE, reduce sample complexity, facilitate detection of less abundant protein components, increases the protein loads and the protein amount in each gel spot for MALDI-MS analysis.     

Proteomic analysis of the endoplasmic reticulum from developing and germinating seed of castor (Ricinus communis)

Endoplasmic reticulum (ER) has been prepared and analysed from germinating and developing castor bean endosperm. A combination of one- and two-dimensional (1-D and 2-D) gel electrophoresis was used to study the complexity of sample and protein differences between the two stages. The ER of the developing oilseed is central to the synthesis, sorting and storage of protein and lipid reserves while the germinating seed is concerned with their degradation. Sample complexity has been reduced by separation of ER proteins into lumenal, peripheral membrane and integral membrane subfractions. Membrane proteins pose specific problems in aggregation and binding to passive surfaces. We have overcome this by collection of membranes at density gradient interfaces and by silanization of plastic ware. Several major components have been identified from 1-D gels by N-terminal sequencing and matrix-assisted laser desorption/ionization (MALDI) peptide mass fingerprints. These include protein disulphide isomerase (PDI), calreticulin and developing-ER-specific oleate-12-hydroxylase involved in the biosynthesis of ricinoleic acid. In excess of 300 spots are detectable in each developmental fraction by high sensitivity 2-D gels. This is the first 2-D electrophoretic analysis of plant ER. These gels reveal significant differences between germinating and developing ER. Preparative loading 2-D gels of germinating ER have been run and 14 selected spots characterized by quadrupole time of flight tandem mass spectrometry (Q-TOF MS/MS). Ten of these proteins were assigned function on the basis of identity with existing castor database entries, or by homology with other species. Two proteins, aspartate proteinase precursor and N-carbamyl-L-aminohydrolase-like protein, appear to be absent from developing profiles. Most of the proteins identified are concerned with roles in protein processing and storage, and lipid metabolism which occur in the ER. Data from three of the assigned spots included unidentified peptides indicating the presence of more than one protein in these spots following 2-D electrophoresis. More extensive analysis will have to await developments in genomics but the basic separation technologies to simplify sample identity for a plant ER preparation have been established.     

The proteome of maize leaves: use of gene sequences and expressed sequence tag data for identification of proteins with peptide mass fingerprints.

As a first step in establishing a proteome database for maize, we have embarked on the identification of the leaf proteins resolved on two-dimensional (2-D) gels. We detected nearly 900 spots on the gels with a pH 4-7 gradient and over 200 spots on the gels with a pH 6-11 gradient when the proteins were visualized with colloidal Coomassie blue. Peptide mass fingerprints for 300 protein spots were obtained with matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometer and 149 protein spots were identified using the protein databases. We also searched the pdbEST databases to identify the leaf proteins and verified 66% of the protein spots that had been identified using the protein databases. Sixty-seven additional protein spots were identified from expressed sequence tags (ESTs). Many abundant leaf proteins are present in multiple spots. Functions of over 50% of the abundant leaf proteins are either unknown or hypothetical. Our results show that EST databases in conjunction with peptide mass fingerprints can be used for identifying proteins from organisms with incomplete genome sequence information.     

Comprehensive proteome analysis of mouse liver by ampholyte-free liquid-phase isoelectric focusing

In this study, ampholyte-free liquid-phase IEF (LIEF) was combined with narrow pH range 2-DE and SDS-PAGE RP-HPLC for comprehensive analysis of mouse liver proteome. Because LIEF prefractionation was able to reduce the complexity of the sample and enhance the loading capacity of IEF strips, the number of visible protein spots on subsequent 2-DE gels was significantly increased. A total of 6271 protein spots were detected after integrating five narrow pH range 2-DE gels following LIEF prefractionation into a single virtual 2-DE gel. Furthermore, the pH 3-5 LIEF fraction and the unfractionated sample were separated by pH 3-6 2-DE and identified by MALDI-TOF/TOF MS, respectively. In parallel, the pH 3-5 LIEF fraction was also analyzed by SDS-PAGE RP-HPLC MS/MS. LIEF-2-DE and LIEF-HPLC could obviously improve the separation efficiency and the confidence of protein identification, which identified a higher number of low-abundance proteins and proteins with extreme physicochemical characteristics or post-translational modifications compared to conventional 2-DE method. Furthermore, there were 207 proteins newly identified in mouse liver in comparison with previously reported large-scale datasets. It was observed that the combination of LIEF-2-DE and LIEF-HPLC was effective in promoting MS-based liver proteome profiling and could be applied on similar complex tissue samples.     

Sample complexity reduction for two-dimensional electrophoresis using solution isoelectric focusing prefractionation

Despite its excellent resolving power, 2-DE is of limited use when analyzing cellular proteomes, especially in differential expression studies. Frequently, fewer than 2000 protein spots are detected on a single 2-D gel (a fraction of the total proteome) regardless of the gel platform, sample, or detection method used. This is due to the vast number of proteins expressed and their equally vast dynamic range. To exploit 2-DE unique ability as both an analytical and a preparative tool, the significant sample prefractionation is necessary. We have used solution isoelectric focusing (sIEF) via the ZOOM IEF Fractionator (Invitrogen) to generate sample fractions from complex bacterial lysates, followed by parallel 2-DE, using narrow-range IPG strips that bracket the sIEF fractions. The net result of this process is a significant enrichment of the bacterial proteome resolved on multiple 2-D gels. After prefractionation, we detected 5525 spots, an approximate 3.5-fold increase over the 1577 spots detected in an unfractionated gel. We concluded that sIEF is an effective means of prefractionation to increase depth of field and improve the analysis of low-abundance proteins.     

Lung alveolar proteomics of bronchoalveolar lavage from a pulmonary alveolar proteinosis patient using high-resolution FTICR mass spectrometry

High-resolution Fourier transform ion cyclotron resonance (FTICR) mass spectrometry was developed and applied to the proteome analysis of bronchoalveolar lavage fluid (BALF) from a patient with pulmonary alveolar proteinosis. With use of 1-D and 2-D gel electrophoresis, surfactant protein A (SP-A) and other surfactant-related lung alveolar proteins were efficiently separated and identified by matrix-assisted laser desorption/ionization FTICR mass spectrometry . Low molecular mass BALF proteins were separated using a gradient 2-D gel. An efficient extraction/precipitation system was developed and used for the enrichment of surfactant proteins. The result of the BALF proteome analysis show the presence of several isoforms of SP-A, in which an N-non-glycosylierte form and several proline hydroxylations were identified. Furthermore, a number of protein spots were found to contain a mixture of proteins unresolved by 2-D gel electrophoresis, illustrating the feasibility of high-resolution mass spectrometry to provide identifications of proteins that remain unseparated in 2-D gels even upon extended pH gradients. Yu Bai and Dmitry Galetskiy both contributed equally to this work.     

Combination of affinity depletion of abundant proteins and reversed-phase fractionation in proteomic analysis of human plasma/serum

The tremendous complexity of the serum and plasma proteome presents extreme analytical challenges in comprehensive analysis due to the wide dynamic range of protein concentrations. Therefore, robust sample preparation methods remain one of the important steps in the proteome characterization workflow. We present the results on a new column for the specific depletion of 14 high-abundant proteins from human serum and plasma and the subsequent reversed-phase fractionation of the flow-through proteins. Analysis of tryptic peptides was accomplished with microfluidic HPLC-Chip/MS system. Results indicate that high-abundant protein depletion combined with RP fractionation of plasma showed an improved dynamic range for proteomic analysis and enabled the identification of low-abundant plasma proteins.     

Identification by mass spectrometry of two-dimensional gel electrophoresis-separated proteins extracted from lager brewing yeast

As two-dimensional (2-D) electrophoresis allows the separation of several hundred proteins in a single gel, this technique has become an important tool for proteome studies and for investigating the cellular physiology. In order to take advantage of information provided by the comparison of proteome pictures, the mass spectrometry technique is the way chosen for a rapid and an accurate identification of proteins of interest. Unfortunately, in the case of industrial yeasts, due to the high level of complexity of their genome, the whole DNA sequence is not yet available and all encoded protein sequences are still unknown. Nevertheless, this study presents here 30 lager brewing yeast proteins newly identified with matrix assisted laser desorption/ionization-time of flight (MALDI-TOF), tandem mass spectrometry (MS/MS) and database searching against the protein sequences of Saccharomyces cerevisiae. The identified proteins of the industrial strain correspond to proteins which do not comigrate with known proteins of S. cerevisiae separated on 2-D gels. This study presents an application of the MS technique for the identification of industrial yeast proteins which are only homologous to the corresponding S. cerevisiae proteins.     

Multi-dimensional HPLC/MS of the nucleolar proteome using HPLC-chip/MS

The proteome of the human nucleolus was investigated in a single analysis using off-line strong cation exchange chromatography and microfraction collection combined with HPLC-chip/MS. The analysis was conducted either as a 1-D workflow with HPLC-chip alone or as a 2-D workflow. Two hundred and six unique proteins were identified in the International Protein Index human database corresponding to 2024 unique tryptic peptides identified in the 2-D analysis. In contrast, only 34 proteins and 151 corresponding tryptic peptides were found by applying a 1-D separation strategy. This clearly indicated that the complexity of the samples required the combination of more than one orthogonal separation technique. Stringent database search criteria, including reversal of sequences and therefore better exclusion of false-positive identifications, were applied for reliable protein identification.     

Recombinant autofluorescent landmarks for standardization of electrophoretic migration of proteins

Unequivocal identification of unknown protein spot patterns in two-dimensional (2-D) electrophoresis still represents a major problem when performing comparative studies of different 2-D electrophoresis gels. Inhomogeneity of gels due to variations in the gel casting procedure, electroendoosmosis and heterogeneity of proteins are major contributions to variations in migration patterns. By fusing green fluorescent protein to a number of well-defined selected proteins (human lysozyme, initiation factor 5a (EIF5a), rapamycin-selective 25 kDa immunophilin (FKBP25), and heat shock protein 90 beta (hsp90)), the isoelectric points and the molecular mass were designed. Proteins were additionally tagged with the FLAG tag enabling rapid purification by immunoaffinity chromatography. The fusion proteins were expressed intracellularly in yeast to avoid heterogeneity caused by post-translational modifications. The quality and applicability was tested in 1-D and 2-D electrophoresis. Sharp bands or symmetric spots were obtained. The proteins are considered as a new generation of reference proteins for electrokinetic separation methods.     

Diagnosis of cellular states of microbial organisms using proteomics.

Two-dimensional (2-D) polyacrylamide gel electrophoresis has much to contribute to experimental analysis of the proteomes of microbial organisms, since this method separates most cellular proteins and allows synthesis rates to be determined quantitatively. Databases generated using 2-D gels can grow to be very large from even just a few experiments, since each sample provides the data for a field (or column) in the database for several hundreds to even thousands of records (or rows), each of which represents a single polypeptide species. The value of such databases for generating an encyclopedia of how each of the cell's proteins behave in different conditions (protein phenotypes) has been recognized for some time. The potential exists, however, to glean even more valuable information from such databases. Because the measurements of each protein are made in the context of all other proteins, a comprehensive glimpse of the cell's physiological state is theoretically achievable with each 2-D gel. By examining enough conditions (and 2-D gels), expression patterns of subsets of proteins (proteomic signatures) can be found that correlate with the cell's state. This type of information can provide a unique contribution to proteomic analysis, and should be a major focus of such analyses.     

Identification of platelet proteins separated by two-dimensional gel electrophoresis and analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry and detection of tyrosine-phosphorylated proteins

Different search programs were compared to judge their particular efficiency in protein identification. We established a human blood platelet protein map and identified tyrosine-phosphorylated proteins. The cytosolic fraction of human blood platelets was separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and phosphorylated proteins were detected by Western blotting using anti-phosphotyrosine antibodies. Visualized protein spots were excised, digested with trypsin and analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The obtained mass fingerprint data sets have been analyzed using ProFound, MS-Fit and Mascot. For those protein spots with no significant search results MALDI post source decay (PSD) spectra have been acquired on the same sample. For automatic interpretation of these fragment ion spectra, the SEQUEST and Mascot algorithm were applied. Another approach for the identification of phosphorylated proteins is immunoprecipitation using an anti-phosphotyrosine antibody. A method for immunoprecipitation of tyrosine-phosphorylated peptides was optimized.     

Knowledge-based proteome profiling: considering identified proteins to evaluate separation efficiency by 2-D PAGE

Proteome profiling techniques rely on the separation of proteins or peptides and their subsequent quantification. The reliability of this technique is still limited because a proteome profiling result does not necessarily represent the true protein composition of the analysed sample, thus seriously hampering proper data interpretation. Many experimentally observed proteome alterations are biologically not significant. It was the aim of this study to use the knowledge of the biological context of proteins in order to establish optimised proteome profiling protocols. While 2-D spot patterns of total cell protein fractions were found to poorly represent the true protein composition, purified subcellular protein fractions were found to better represent the protein composition of the analysed sample. The application of a standardised protocol to different kinds of cells revealed several striking observations. Firstly, the protein composition of cultured cells of various origins is very similar. Secondly, proteome alterations observed with the described protocols do make sense from a biologic point of view and may thus be considered as truly representative for the analysed samples. Thirdly, primary white blood cells isolated from different donors were found to show minor, but reproducible and significant individual differences. We designate the consideration of known properties of identified proteins in proteome profiles as a knowledge-based approach. The present data suggest that this approach may tremendously help to improve the applied techniques and assess the results. We demonstrate that the fulfilment of well-defined criteria of proteome profiles eventually results in reliable and biologically relevant data.     

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.