Profiling of Caenorhabditis elegans proteins using two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-time of flight-mass spectrometry

The nematode Caenorhabditis elegans (C. elegans) is the first animal whose whole 97 Mb genome sequence, encoding ca. 19000 open reading frames (ORF's), has been essentially determined. We tried to establish a 2-DE map of the nematode proteome by means of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). A soluble protein fraction of mixed stages of the worm, wild-type strain N2, was applied to 2-D PAGE. After Coomassie Brilliant Blue (CBB) staining, 1200 spots were detected and 140 major spots were excised from the gel and subjected to in-gel digestion with Achromobacter protease I (lysyl endopeptidase). Resulting peptides were analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) followed by peptide mass fingerprinting for protein identification. With this approach we have obtained a two-dimensional electrophoresis (2-DE) protein map in which 69 spots were localized as landmarks for comparison of expression profiles to elucidate the basis of various biological events.     

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.     

Impact of deglycosylation methods on two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-time of flight-mass spectrometry for proteomic analysis

Glycosylation is a common post-translational modification that can add complexity to the proteome of many cell types. We used enzymatic and chemical methods of deglycosylation to treat a heavily glycosylated exoproteome sample from the filamentous fungus Trichoderma reesei. Deglycosylated samples were resolved on one-dimensional (1-D) and two-dimensional (2-D) gels in order to determine the effect of deglycosylation on the electrophoresis patterns and on the ability to identify proteins by peptide mass matching using matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis of in-gel tryptic digests. We found that deglycosylation of the protein sample resulted in different protein patterns on 1-D and 2-D gels, reduced the complexity of gel patterns, and enhanced the protein identification of some proteins via MALDI-TOF-MS. Deglycosylation with trifluoromethanesulfonic acid (TFMS) was found to be more effective than enzymatic treatments. These deglycosylation techniques may be employed in whole proteome analysis to locate glycosylated proteins and assist in their identification by MS.     

Analysis of cellular release using capillary electrophoresis and matrix assisted laser desorption/ionization-time of flight-mass spectrometry.

In order to increase our understanding of the mechanisms of learning and memory in the central nervous system, it is necessary to know the neurotransmitters and neuromodulators used in the specific neuronal circuits under study. Methods have been developed to identify the peptides released from single neurons and neuronal clusters from the common neuronal model Aplysia californica. Specifically, solid-phase extraction (SPE), capillary electrophoresis (CE) and matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) are combined for profiling neuropeptide releasates. A variety of combinations of SPE and CE were coupled off-line with MALDI-TOF-MS to reduce the high physiological salts, to concentrate the analytes, and to reduce the complexity of the mass spectra using separation. With these protocols, peptides and proteins up to 11000 Da were detected in releasates, offering a much wider mass range compared to direct MALDI analysis of the same releasates. A number of expected and unknown neuropeptides, including egg-laying hormone (ELH) and the partially processed delta/gamma-bag cell peptide were observed in the SPE-treated releasates from a single Aplysia-cultured bag cell neuron. However, by adding a CE separation after the SPE step preceding off-line MALDI-TOF-MS detection, the most complete neuropeptide profiles were obtained.     

Identification of the apolipoprotein E4 isoform in cerebrospinal fluid with preparative two-dimensional electrophoresis and matrix assisted laser desorption/ionization-time of flight-mass spectrometry.

Apolipoprotein E (apoE) was isolated from human cerebrospinal fluid (CSF) from control individuals and patients with Alzheimer's disease (AD). The purification was performed with preparative two-dimensional electrophoresis (2-DE), involving liquid-phase isoelectric focusing (IEF) in the Rotofor cell in combination with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electroelution in the Mini Whole Gel Eluter. ApoE was characterized by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis of tryptic digests. The known change of Cys to Arg in position 112 of the apoE4 isoform was identified. This was detected in CSF from AD patients, reflecting the increased frequency of the apoE4 allele in this population. This peptide was not detected in CSF samples from healty control individuals. The use of this rapid electrophoretic separation in proteomic studies of CSF proteins provides single proteins, such as apoE, of high purity in yields sufficient for characterization by MALDI-TOF-MS. Characterization of proteins and their modifications (amino acid substitutions, glycosylation or phosphorylation) in CSF will be a useful tool in the investigation of the pathophysiology of brain disorders such as AD.     

Sequence information of peptides and proteins with in-source decay in matrix assisted laser desorption/ionization-time of flight-mass spectrometry

In-source decay coupled with matrix assisted laser desorption/ionization-mass spectrometry, which is a mass spectrometric degradation method for the sequencing of peptides and proteins, has been applied to several different polypeptides and proteins. The influence of the nature of the constituent amino acids on positively charged product ions is described. Relatively small molecular mass peptides produced c-, b-, and/or a-series ions usable for C-terminal sequencing as well as y- and/or z-series ions usable for N-terminal sequencing. The formation of the C-terminal sequencing ions (c, b and a) and the N-terminal sequencing ions (y and z) was strongly dependent on the location(s) of basic arginine and lysine residues. The presence of the arginine and/or lysine residues at the N-terminal region was one-sided in the formation of c-, b-, and/or a-series ions, while the presence of those at the C-terminal region was favorable for the formation of y- and z-series ions. In-source decay experiments of intact proteins, apomyoglobin and two viral coat proteins, led to large amounts of c-series ions and small amounts of y-series ions, which reflected internal sequences.     

Immobilized metal-ion chelating capillary microreactor for peptide mapping analysis of proteins by matrix assisted laser desorption/ ionization-time of flight-mass spectrometry

Peptide mass mapping analysis, utilizing a regenerable enzyme microreactor with metal-ion chelated adsorption of enzyme, combined with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) was developed. Different procedures from the conventional approaches were adopted to immobilize the chelator onto the silica supports, that is, the metal chelating agent of iminodiacetic acid (IDA) was reacted with glycidoxypropyltrimethoxysilane (GLYMO) before its immobilization onto the inner wall of the fused-silica capillary pretreated with NH(4)HF(2). The metal ion of copper and subsequently enzyme was specifically adsorbed onto the surface to form the immobilized enzyme capillary microreactor, which was combined with MALDI-TOF-MS to apply for the mass mapping analysis of nL amounts of protein samples. The results revealed that the peptide mapping could routinely be generated from 0.5 pmol protein sample in 15 min at 50 degrees C, even 20 fmol cytochrome c could be well digested and detected.     

Droplet electrocoupling between capillary electrophoresis and matrix assisted laser desorption/ionization-time of flight-mass spectroscopy and its application.

A droplet of an electroconductive solution was put on the sample plate of a matrix-assisted laser desorption/ionization-time of flight-mass spectroscope (MALDI-TOF-MS) and the outlet terminal of a capillary Electrophoresis (CE) capillary was put into this droplet in order to make an electro-connection and to apply high voltage between the metallic sample plate and the counter pole of the CE. This procedure was simple and gave much more stable interfacing than that of the electrospray method. Furthermore, the separated component was collected and concentrated in a droplet. By mixing each separated sample spot with the MALDI matrix on the sample plate, the spots were analyzed in separated sequences to make three-dimensional mass chromatograms, or applied to the enzyme digestion analysis for peptide sequencing.     

Alkylation power of free Immobiline chemicals towards proteins in isoelectric focusing and two-dimensional maps, as explored by matrix assisted laser desorption/ionization-time of flight-mass spectrometry

A number of Immobilines, with pK 1.0-10.3, were incubated with two proteins, bovine alpha-lactalbumin (pI 4.80) and chicken egg lysozyme (pI 9.32), at pH approximately 9-10 and the resulting solutions were examined by matrix assisted laser desorption/ionization mass spectrometry. The reflectron mode of the same technique was also used to analyze a number of tryptic digests of some solutions. The extent and the number of detected alkylation sites associated with the acidic protein were found to be linearly proportional to the pK values of the investigated Immobilines, an effect which was less evident for the basic protein. The high resolution measurements of some tryptic digests indicate the cysteine residues as the likely sites of alkylation. The implications of the present data for isoelectric focusing separations on immobilized pH gradients and for two-dimensional maps are discussed.     

Recent advancements in surface-enhanced laser desorption/ionization-time of flight-mass spectrometry

The overall history and recent advances in surface enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS) technology is reviewed herein. Fundamentals of SELDI-TOF analysis are presented while drawing comparisons with other laser-based mass spectrometry techniques. The application of SELDI-TOF-MS to functional genomics and biomarker discovery is discussed and exemplified by elucidating a biomarker candidate for prostatic carcinoma. Finally, a short discussion regarding future SELDI requirements and developments is supplied.     

Large-gel two-dimensional electrophoresis-matrix assisted laser desorption/ionization-time of flight-mass spectrometry: an analytical challenge for studying complex protein mixtures

The large-gel two-dimensional electrophoresis (2-DE) technique, developed by Klose and co-workers over the past 25 years, provides the resolving power necessary to separate crude proteome extracts of higher eukaryotes. Matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) provides the sample throughput necessary to identify thousands of different protein species in an adequate time period. Spot excision, in situ proteolysis, and extraction of the cleavage products from the gel matrix, peptide purification and concentration as well as the mass spectrometric sample preparation are the crucial steps that interface the two analytical techniques. Today, these routines and not the mass spectrometric instrumentation determine how many protein digests can be analyzed per day per instrument. The present paper focuses on this analytical interface and reports on an integrated protocol and technology developed in our laboratory. Automated identification of proteins in sequence databases by mass spectrometric peptide mapping requires a powerful search engine that makes full use of the information contained in the experimental data, and scores the search results accordingly. This challenge is heading a second part of the paper.     

Separation and first structure elucidation of Cremophor EL-components by hyphenated capillary electrophoresis and delayed extraction-matrix assisted laser desorption/ionization-time of flight-mass spectrometry

The polyethoxylated heterogeneous components of the so far poorly characterized nonionic emulsifier Cremophor EL (polyoxyl 35 castor oil) (CrEL) were fractionated by cyclodextrin-modified micellar electrokinetic capillary chromatography (CD-MEKC). Due to the low UV absorbance of most of the CrEL-components an indirect UV detection was used with phenobarbital-sodium as background absorber. For a precise assignment of the resulting peaks to the corresponding components capillary electrophoresis (CE) had to be combined with delayed extraction-matrix assisted laser desorption/ionization-time of flight-mass spectrometry (DE-MALDI-TOF-MS) as detection system. For this purpose, the fractionating robot Probot was employed which enables both the on-line fractionation of the CE eluate on a MALDI target during the electrophoretic separation and the simultaneous dosage of the MALDI matrix solution. The applied CrEL amount was optimized by varying the CE injection parameters time, pressure and concentration of the sample in order to obtain homologue peak series of sufficient intensity without decreasing the separation efficiency. Evaluation of the mass spectra was performed by comparing the residue masses of the homologue peak series with the calculated residue masses of potential CrEL-components. However, the high number of polyethoxylated components leads to overlapping of homologue peak series with isobaric residue masses. These isobaric interferences were detected by a high mass accuracy of the measurements (obtained by internal calibration with polyethylene glycol (PEG) 1000 and by means of the residue mass plot, the newly developed evaluation method. The combination of these techniques allowed the first detailed structure analysis of the CrEL-components showing glycerol polyoxyethylene (POE) monoricinoleate and POE monoricinoleate to be the two main components of the emulsifier. Furthermore, the coupling of CE with DE-MALDI-TOF-MS is generally applicable to the fractionation and identification of polymers.     

Proteome analysis of human liver tumor tissue by two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-mass spectrometry for identification of disease-related proteins

Hepatocellular carcinoma (HCC) is a common malignancy worldwide and is a leading cause of death. To contribute to the development and improvement of molecular markers for diagnostics and prognostics and of therapeutic targets for the disease, we have largely expanded the currently available human liver tissue maps and studied the differential expression of proteins in normal and cancer tissues. Reference two-dimensional electrophoresis (2-DE) maps of human liver tumor tissue include labeled 2-DE images for total homogenate and soluble fraction separated on pH 3-10 gels, and also images for soluble fraction separated on pH 4-7 and pH 6-9 gels for a more detailed map. Proteins were separated in the first dimension by isoelectric focusing on immobilized pH gradient (IPG) strips, and by 7.5-17.5% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels in the second dimension. Protein identification was done by peptide mass fingerprinting with delayed extraction-matrix assisted laser desorption/ionization-time of flight-mass spectrometry (DE-MALDI-TOF-MS). In total, 212 protein spots (117 spots in pH 4-7 map and 95 spots in pH 6-9) corresponding to 127 different polypeptide chains were identified. In the next step, we analyzed the differential protein expression of liver tumor samples, to find out candidates for liver cancer-associated proteins. Matched pairs of tissues from 11 liver cancer patients were analyzed for their 2-DE profiles. Protein expression was comparatively analyzed by use of image analysis software. Proteins whose expression levels were different by more than three-fold in at least 30% (four) of the patients were further analyzed. Numbers of protein spots overexpressed or underexpressed in tumor tissues as compared with nontumorous regions were 9 and 28, respectively. Among these 37 spots, 1 overexpressed and 15 underexpressed spots, corresponding to 11 proteins, were identified. The physiological significance of the differential expressions is discussed.     

Proteomic analysis of differential protein expression in a human hepatoma revertant cell line by using an improved two-dimensional electrophoresis procedure combined with matrix assisted laser desorption/ionization-time of flight-mass spectrometry

The proteomic profiles of a human hepatoma revertant, CL1, and its original cell line, SMMC7721, were compared by using an improved two-dimensional electrophoresis (2-DE) procedure, with multi-IPGstrips gels (length 相似文献   

Identification of two-dimensionally separated human cerebrospinal fluid proteins by N-terminal sequencing, matrix-assisted laser desorption/ionization--mass spectrometry, nanoliquid chromatography-electrospray ionization-time of flight-mass spectrometry, and tandem mass spectrometry

Optimal application of biological mass spectrometry (MS) in combination with two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) of human cerebrospinal fluid (CSF) can lead to the identification of new potential biological markers of neurological disorders. To this end, we analyzed a number of 2-D PAGE protein spots in a human CSF pool using spot co-localization, N-terminal sequencing, matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and nanoliquid chromatography-electrospray ionization-time of flight-mass spectrometry (nanoLC-ESI-TOF-MS) with tandem MS switching. Our constructed CSF master contained 469 spots after image analysis and processing of 2-D gels. Upon visual inspection of our CSF master with the CSF pattern available on the ExPASy server, it was possible to locate and annotate 15 proteins. N-terminal sequence analysis and MALDI-MS peptide mass fingerprint analysis of both silver- and Coomassie Brilliant Blue (CBB) G-250-stained protein spots after in situ trypsin digest not only confirmed nine of the visually annotated spots but additionally resolved the identity of another 13 spots. Six of these proteins were not annotated on the 2-D ExPASy map: complement C3 alpha-chain (1321-1663), complement factor B, cystatin C, calgranulin A, hemoglobin beta-chain, and beta-2-microglobulin. It was clear that MALDI-MS identification from CBB G-250-stained, rather than from silver-stained, spots was more successful. In cases where no N-terminal sequence and/or no clear MALDI-MS result was available, nanoLC-ESI-TOF-MS and tandem MS automated switching was used to clarify and/or identify these protein spots by generating amino acid sequence tags. In addition, enrichment of the concentration of low-abundant proteins on 2-D PAGE was obtained by removal of albumin and immunoglobulins from the CSF pool using affinity chromatography. Subsequent analysis by 2-D PAGE of the fractionated CSF pool showed various new silver-stainable protein spots, of which four were identified by nanoLC-ESI-TOF-MS and tandem MS switching. No significant homology was found in either protein or DNA databases, indicating than these spots were unknown proteins.     

Classification of wheat varieties: use of two-dimensional gel electrophoresis for varieties that can not be classified by matrix assisted laser desorpiton/ionization-time of flight-mass spectrometry and an artificial neural network

Analyzing a gliadin extract by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) combined with an artificial neural network (ANN) is a suitable method for identification of wheat varieties. However, the ANN can not distinguish between all different wheat varieties. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was applied to three pairs of wheat varieties, which can not be classified correctly by ANN. By 2-D PAGE the varieties in the three pairs can be discriminated and these six wheat varieties can be separated from each other, which could not be separated by MALDI-TOF-MS and NN.     

Assessing matrix assisted laser desorption/ ionization-time of flight-mass spectrometry as a means of rapid embryo protein identification in rice.

Rice embryo proteins were separated by two-dimensional gel electrophoresis (2-DE). A total of 105 spots were digested with trypsin and the resultant peptides were analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Raw mass spectra were fully-automatically processed and searched with selected monoisotopic masses against SWISS-PROT/TrEMBL and NCBInr databases. High quality mass spectra were obtained from 53 spots, of which 36 spots were identified including 29 not registered in databases. Fifty percent of the rice embryo proteins resolved in 2-DE could not be identified, indicating more efficient sample preparation techniques need to be developed in the future. At least four to five matching peptides were found to be essential for unambiguous identification of rice embryo proteins; peptide matching of less than four lead to ambiguous results. The suitability of peptide mass fingerprinting method as a means of rapid embryo protein identification in rice was discussed.     

Disposable polymeric high-density nanovial arrays for matrix assisted laser desorption/ionization-time of flight-mass spectrometry: II. Biological applications.

A novel disposable high-density matrix assisted laser desorption/ionization (MALDI) target plate made either from polymethylmethacrylate (PMMA) or polycarbonate (PC) is presented where thousands (1,200-1,600) of samples can be deposited and subsequently analyzed by MALDI-time of flight (TOF) mass spectrometry. Good reproducibility was obtained across the plate regardless of position on the target plate with a relative standard deviation (RSD) on the peak intensity of typically 30% calculated from data generated by analysis of a 10 nm peptide mixture of angiotensin I, II, III and bradykinin. The nanovial array format combined with microdispensing technology makes it possible to carry out in-vial chemistry on deposited samples. This is demonstrated by the analysis of peptides from beta-casein and subsequent in-vial dephosphorylation of its phosphopeptides at 10 fmol levels by microdispensing of alkaline phosphatase, into the nanovial. The mass spectra obtained from these polymeric targets provides can also be used in high sensitivity applications as shown by peptide mass fingerprinting of human fibroblast proteins separated by two-dimensional gel electrophoresis.     

Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry of lipopolysaccharide species separated by slab-polyacrylamide gel electrophoresis: high-resolution separation and molecular weight determination of lipooligosaccharides from Vibrio fischeri strain HMK

We recently demonstrated that the combined use of lipopolysaccharide (LPS) reverse staining and high-efficiency passive elution techniques can be successfully used as a suitable interface between LPS slab-gel separation and electrospray ionization-mass spectrometry (ESI-MS) of LPS-derived oligosaccharides. Here, we extend our micropurification strategy for the analysis of O-deacylated LPS forms from Vibrio fischeri HMK after recovery from single reverse-stained LPS bands using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The quantities (30-40 microg) obtained from the two gel-resolved LPS bands were sufficient to allow MALDI-TOF-MS detection of O-deacylated LPS glycoforms at m/z 3767.1, 3890.1 for the high-molecular-weight or at m/z 2522.5, 2645.4, 2725.7, and 2848.7 for the low-molecular-weight LPS band. These LPS band heterogeneities resulted not only from variations in the oligosaccharide region of the LPS but also from two phosphorylation states of the lipid A (diphosphoryl and diphosphoryl plus a single phosphoethanolamine substitution). On the other hand, MALDI-TOF mass spectra of the separated LPS bands displayed reduced heterogeneity and increased signal-to-noise ratios as compared to spectra of the unpurified LPS. Furthermore, micropurification of LPS bands prior MALDI-TOF-MS led to a higher sensitivity of detection of less abundant low-molecular-weight LPS glycoforms. Taken together, this and our previous study on gel-micropurified LPS using ESI definitively show how one can unambiguously determine the different molecular species contained within each gel-separated LPS band, their relative abundance and oligosaccharide sequences.     

Disposable polymeric high-density nanovial arrays for matrix assisted laser desorption/ionization-time of flight-mass spectrometry: I. Microstructure development and manufacturing.

In order to meet the expected enormous demand for mass spectrometry (MS) throughput as a result of the current efforts to completely map the human proteome, this paper presents a new concept for low-cost high-throughput protein identification by matrix assisted laser desorption/ionization-time of flight-(MALDI-TOF)-MS peptide mapping using disposable polymeric high-density nanovial MALDI target plates. By means of microfabrication technology precision engineered nanovial arrays are fabricated in polymer substrates such as polymethylmethacrylate (PMMA) and polycarbonate (PC). The target plate fabrication processes investigated were precision micromilling, cold embossing and injection moulding (work in progress). Nanovial dimensions were 300, 400 or 500 microm. Typical array densities were 165 nanovials/cm2, which corresponds to 3,300 vials on a full Applied Biosystems MALDI target plate. Obtained MALDI data displayed equal mass resolution, accuracy, signal intensity for peptide standards as compared to high-density silicon nanovial arrays previously reported by our group [7], as well as conventional stainless steel or gold targets.