Microwave-assisted protein preparation and enzymatic digestion in proteomics

The combinations of gel electrophoresis or LC and mass spectrometry are two popular approaches for large scale protein identification. However, the throughput of both approaches is limited by the speed of the protein digestion process. Present research into fast protein enzymatic digestion has been focused mainly on known proteins, and it is unclear whether these results can be extrapolated to complex protein mixtures. In this study microwave technology was used to develop a fast protein preparation and enzymatic digestion method for protein mixtures. The protein mixtures in solution or in gel were prepared and digested by microwave-assisted protein enzymatic digestion, which rapidly produces peptide fragments. The peptide fragments were further analyzed by capillary LC and ESI-ion trap-MS or MALDI-TOF-MS. The technique was optimized using bovine serum albumin and then applied to human urinary proteins and yeast lysate. The method enabled preparation and digestion of protein mixtures in solution (human urinary proteins) or in gel (yeast lysate) in 6 or 25 min, respectively. Equivalent (in-solution) or better (in-gel) digestion efficiency was obtained using microwave-assisted protein enzymatic digestion compared with the standard overnight digestion method. This new application of microwave technology to protein mixture preparation and enzymatic digestion will hasten the application of proteomic techniques to biological and clinical research.     

Proteins isolated with TRIzol are compatible with two-dimensional electrophoresis and mass spectrometry analyses

TRIzol is used for RNA isolation but also permits protein recovery. We investigated whether proteins prepared with TRIzol were suitable for two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization mass spectrometry. Proteins from TRIzol-treated SH-SY5Y cells produced 2-DE spot patterns similar to those from an equivalent untreated sample. Subsequent identification of TRIzol-treated proteins using peptide mass fingerprinting was successful. TRIzol exposure altered neither the mass of myoglobin extracted from sodium dodecyl sulfate (SDS) gels nor the masses of myoglobin peptides produced by in-gel trypsin digestion. These findings suggest that proteins isolated with TRIzol remain amenable to proteomic analyses.     

Utility of dry gel from two-dimensional electrophoresis for peptide mass fingerprinting analysis of silkworm proteins

We compared the use of wet and dry two-dimensional electrophoresis (2-DE) gels for in-gel tryptic digestion and subsequent analysis by mass spectrometry, first using bovine serum albumin (BSA) as a model protein and then using unknown proteins from an extract of the silkworm midgut. The gel was either dried at 80 degrees C or left wet. Upon analysis of BSA, there was little difference in peptide recovery from 2-DE or in mass spectrum between the dry and the wet gels. The midgut extract was resolved into more than 1,100 protein spots by 2-DE, and 40 of these spots were sampled for further analysis. For all of the 40 proteins, the results obtained from dry and wet gels were quite similar in mass spectra and protein identification, although the relative amounts of peptides from tryptic digestion ranged from 45 to 146%. Based on these results, we confirmed the utility of dry electrophoretic gels for proteomics of insect extracts.     

Proteome analysis of Escherichia coli using high-performance liquid chromatography and Fourier transform ion cyclotron resonance mass spectrometry

The basic problem of complexity poses a significant challenge for proteomic studies. To date two-dimensional gel electrophoresis (2-DE) followed by enzymatic in-gel digestion of the peptides, and subsequent identification by mass spectrometry (MS) is the most commonly used method to analyze complex protein mixtures. However, 2-DE is a slow and labor-intensive technique, which is not able to resolve all proteins of a proteome. To overcome these limitations gel-free approaches are developed based on high performance liquid chromatography (HPLC) and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). The high resolution and excellent mass accuracy of FT-ICR MS provides a basis for simultaneous analysis of numerous compounds. In the present study, a small protein subfraction of an Escherichia coli cell lysate was prepared by size-exclusion chromatography and proteins were analyzed using C4 reversed phase (RP)-HPLC for pre-separation followed by C18 RP nanoHPLC/nanoESI FT-ICR MS for analysis of the peptide mixtures after tryptic digestion of the protein fractions. We identified 231 proteins and thus demonstrated that a combination of two RP separation steps - one on the protein and one on the peptide level - in combination with high-resolution FT-ICR MS has the potential to become a powerful method for global proteomics studies.     

Matrix-assisted laser desorption–ionization mass spectrometry peptide mass fingerprinting for proteome analysis: identification efficiency after on-blot or in-gel digestion with and without desalting procedures

In theory, peptide mass fingerprinting by matrix assisted laser desorption–ionization mass spectrometry (MALDI-MS) has the potential to identify all of the proteins detected by silver staining on gels. In practice, if the genome of the organism investigated is completely sequenced, using current techniques, all proteins stained by Coomassie Brilliant Blue can be identified. This loss of identification sensitivity of ten to hundred-fold is caused by loss of peptides by surface contacts. Therefore, we performed digestion and transfer of peptides in the lower μl range and reduced the number of steps. The peptide mix obtained from in-gel or on-blot digestion was analyzed directly after digestion or after concentration on POROS R2 beads. Eight protein spots of a 2-DE gel from Mycobacterium bovis BCG were identified using these four preparation procedures for MALDI-MS. Overall, on-blot digestion was as effective as in-gel digestion. Whereas higher signal intensities resulted after concentration, hydrophilic peptides are better detected by direct measurement of the peptide mix without POROS R2 concentration.     

Towards global analysis of mosquito chorion proteins through sequential extraction, two-dimensional electrophoresis and mass spectrometry

This study describes the separation and identification of chorion proteins through two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) techniques. Due to their high hydrophobicity, chorion proteins are difficult to be solubilized and absorbed into the immobilized pH gradient strip for isoelectric focusing. By optimizing the applied conditions for chorion protein extraction and sample application, we were able to solubilize the majority of the chorion proteins and resolve them by 2-DE. Under optimized conditions, there are more than 700 protein spots resolved by 2-D analysis. Trypsin digestions of individual protein spots, MALDI-TOF MS analysis of their digested peptides, and subsequent BLAST search of peptide masses resulted in the tentative identification of 38 protein spots. Our data show that sequential extraction of the isolated chorion, 2-DE of the solubilized chorion proteins, in-gel digestion of the resolved protein and MALDI-TOF MS analysis of the protein digests is an effective overall strategy towards determination of chorion proteins in mosquitoes. The merits of the method described for the determination of mosquito chorion proteins and its feasibility for the separation and identification of membrane proteins and chorion or eggshell proteins from other insect species are discussed.     

On-chip microextraction for proteomic sample preparation of in-gel digests

Despite the high sensitivity and relatively high tolerance for contaminants of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) there is often a need to purify and concentrate the sample solution, especially after in-gel digestion of proteins separated by two-dimensional gel electrophoresis (2-DE). A silicon microextraction chip (SMEC) for sample clean-up and trace enrichment of peptides was manufactured and investigated. The microchip structure was used to trap reversed-phase chromatography media (POROS R2 beads) that facilitates sample purification/enrichment of contaminated and dilute samples prior to the MALDI-TOF MS analysis. The validity of the SMEC sample preparation technique was successfully investigated by performing analysis on a 10 nM peptide mixture containing 2 m urea in 0.1 m phosphate-buffered saline with MALDI-TOF MS. It is demonstrated that the microchip sample clean-up and enrichment of peptides can facilitate identification of proteins from 2-DE separations. The microchip structure was also used to trap beads immobilized with trypsin, thereby effectively becoming a microreactor for enzymatic digestion of proteins. This microreactor was used to generate a peptide map from a 100 nM bovine serum albumin sample.     

Microwave-assisted specific chemical digestion for rapid protein identification

We have developed a rapid microwave-assisted protein digestion technique based on classic acid hydrolysis reaction with 2% formic acid solution. In this mild chemical environment, proteins are hydrolyzed to peptides, which can be directly analyzed by MALDI-MS or ESI-MS without prior sample purification. Dilute formic acid cleaves proteins specifically at the C-terminal of aspartyl (Asp) residues within 10 min of exposure to microwave irradiation. By adjusting the irradiation time, we found that the extent of protein fragmentation can be controlled, as shown by the single fragmentation of myoglobin at the C-terminal of any of the Asp residues. The efficacy and simplicity of this technique for protein identification are demonstrated by the peptide mass maps of in-gel digested myoglobin and BSA, as well as proteins isolated from Escherichia coli K12 cells.     

The synaptic vesicle proteome: a comparative study in membrane protein identification

A proteomic analysis of the synaptic vesicle was undertaken to obtain a better understanding of vesicle regulation. Synaptic vesicles primarily consist of integral membrane proteins that are not well resolved on traditional isoelectric focusing/two-dimensional gel electrophoresis (IEF/2-DE) gels and are resistant to in-gel digestion with trypsin thereby reducing the number of peptides available for mass spectrometric analysis. To address these limitations, two complementary 2-DE methods were investigated in the proteome analysis: (a) IEF/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (IEF/SDS-PAGE) for resolution of soluble proteins and, (b) Benzyl hexadecyl ammonium chloride/SDS-PAGE (16-BAC/SDS-PAGE) for resolution of integral membrane proteins. The IEF/SDS-PAGE method provided superior resolution of soluble proteins, but could only resolve membrane proteins with a single transmembrane domain. The 16-BAC/SDS-PAGE method improved separation, resolution and identification of integral membrane proteins with up to 12 transmembrane domains. Trypsin digestion of the integral membrane proteins was poor and fewer peptides were identified from these proteins. Analysis of both the peptide mass fingerprint and the tandem mass spectra using electrospray ionization quadrupole-time of flight mass spectrometry led to the positive identification of integral membrane proteins. Using both 2-DE separation methods, a total of 36 proteins were identified including seven integral membrane proteins, 17 vesicle regulatory proteins and four proteins whose function in vesicles is not yet known.     

Sequencing of rat liver cytosolic proteins by matrix-assisted laser desorption ionization-quadrupole time-of-flight mass spectrometry following electrophoretic separation and extraction

A new technique is described that enables the direct determination of the complete or partial amino acid sequence of cytosolic proteins separated by gel electrophoresis and allows for the further observation of disease- or drug-induced posttranslational modifications. The procedure uses a two-phase extraction strategy (ethyl acetate/ammonium bicarbonate) for the efficient separation of proteins/peptides from an electrophoretic matrix and subsequent sequence analysis by matrix-assisted laser desorption ionization-quadrupole time-of-flight mass spectrometry. The method was tested using hepatocyte cytosolic proteins and compared to a complementary approach using direct solvent extraction from in-gel digests. Although the latter procedure identified the proteins, it did not enable complete amino acid sequence determination. In contrast, high sequence coverage was obtained using the peptide extraction procedure, without any apparent dependence on protein size. The technique minimized the chemically inconsistent modifications generated from in-gel digestion, thus aiding mass spectrometric interpretation and valid protein sequence identification.     

橡胶树死皮病胶乳C-乳清差异表达蛋白质的筛选与鉴定

橡胶树死皮病(Tapping Panel Dryness,TPD)在世界各橡胶种植园普遍发生,给橡胶种植业带来严重的危害。为了更好地了解和阐明死皮病发生、发展的分子机制,本研究应用双向凝胶电泳技术（2-DE）比较橡胶树死皮株与健康株胶乳C-乳清蛋白质组表达的差异。采用固相pH梯度双向凝胶电泳分离橡胶树死皮株与健康株C-乳清的总蛋白质,凝胶经考染显色后,用PDQuest7.40图像分析软件进行比较分析,识别差异表达的蛋白质。这些点经过胶内酶切后进行基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)分析获取肽质指纹图谱(PMF),Mascot软件搜索SWISS-PROT和NCBInr数据库鉴定蛋白质。结果：①橡胶树死皮株与健康株C-乳清凝胶的平均蛋白质点数分别为1075±35和1134±27,其平均匹配的点数分别为982±38和1008±22,组内图像匹配率达91.89﹪和88.72﹪。②橡胶树死皮株与健康株C-乳清组间的平均匹配蛋白点数为970±25。利用MALDI-TOF-MS质谱技术对40个差异明显的蛋白点进行分析鉴定,通过查询数据库鉴定了27个蛋白质。本研究建立了分辨率高且重复性较好的橡胶树死皮株与 健康株胶乳C-乳清的双向凝胶电泳图谱,并应用质谱技术鉴定了其中表达差异的蛋白质点,这些差异表达的蛋白质可能参与了死皮发生和发展的过程。     

Identification of Proteins by Matrix-Assisted Laser Desorption Ionization–Mass Spectrometry Following In-Gel Digestion in Low-Salt, Nonvolatile Buffer and Simplified Peptide Recovery

Matrix-assisted laser desorption ionization–mass spectrometry is an efficient analytical method for large-scale identification of proteins separated by two-dimensional polyacrylamide gel electrophoresis. Following in-gel digestion, the salt present in the peptide extracts is usually removed by chromatography prior to analysis. Desalting is a labor-intensive and time-consuming step, limiting the total number of samples that can be processed daily. We improved the daily sample output by performing the in-gel protein digestion in low-salt, nonvolatile buffer and simplifying the recovery of the generated peptides, collecting them in a small volume by sonication. This technique is routinely used for identification of proteins ofHaemophilus influenzaeand human brain. The methodology described facilitates the analytical process and allows the analysis of hundreds of proteins per day. Furthermore, it represents an essential step toward process automation.     

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.     

Improvement of an in-gel tryptic digestion method for matrix-assisted laser desorption/ionization-time of flight mass spectrometry peptide mapping by use of volatile solubilizing agents

The combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), in-gel enzymatic digestion of proteins separated by two-dimensional gel electrophoresis and searches of molecular weight in peptide-mass databases is a powerful and well established method for protein identification in proteomics analysis. For successful protein identification by MALDI-TOF mass spectrometry of peptide mixtures, critical parameters include highly specific enzymatic cleavage, high mass accuracy and sufficient numbers and sequence coverage of the peptides which can be analyzed. For in-gel digestion with trypsin, the method employed should be compatible both with enzymatic cleavage and subsequent MALDI-TOF MS analysis. We report here an improved method for preparation of peptides for MALDI-TOF MS mass fingerprinting by using volatile solubilizing agents during the in-gel digestion procedure. Our study clearly demonstrates that modification of the in-gel digestion protocols by addition of dimethyl formamide (DMF) or a mixture of DMF/N,N-dimethyl acetamide at various concentrations can significantly increase the recovery of peptides. These higher yields of peptides resulted in more effective protein identification.     

Standardisation of rapid in-gel digestion by mass spectrometry

In-gel digestion has been standardised using a poly(propylene) disposable. We designed a four-step rapid and simple in-gel digestion protocol which is carried out in one self-contained reaction tube avoiding keratin contamination. In order to quantify the efficiency of in-gel digestion, we developed a rapid on-column peptide acetylation protocol. Results show that trypsin in-gel uptake is increased and in-gel digestion is 90% complete within 15 min. We further show that spectrum quality, peptide yield and sequence coverage for mass spectrometric analysis are enhanced. We utilise 2-D PAGE separation of photosystem II from barley to demonstrate that the protocol facilitates identification of highly hydrophobic membrane proteins.     

An optimized protein in-gel digest method for reliable proteome characterization by MALDI-TOF-MS analysis

Human lung epithelial cells (A549) were used as a model to develop a reliable proteome characterization method by peptide mass fingerprinting (PMF). Lung cell lysate proteins and protein standards were separated by 2D-gel electrophoresis, stained with Coomassie blue, gel plugs were subjected to commonly adapted as well as optimized in-gel digestion/sample preparation methods. Samples were analyzed by MALDI-TOF-MS. Optimization parameters included, use of NH(4)OAc in destaining and in-gel digestion buffers, detergent/salt removal prior to in-gel digestion, use of solvents of varying polarities (0%, 30%, 60% ACN containing 0.1% TFA) to improve peptide recoveries, matrix composition (alpha-cyano-4-hydroxycinamic acid-organic solvent combinations) and on-target salt removal. This led to enhanced mass spectral information and a sensitivity gain in the order of 6-10 fold compared to that of common procedures, yielding reliable, unambiguous protein identification with femtomol protein sensitivity by Autoflex MALDI-TOF-MS. Triplicate analyses by two analysts revealed consistent, wide range m/z values including in < 1200Da region by relieving matrix-exerted signal suppression, requiring one trial to obtain a unique protein identification with superior PMF results for the optimized method. Analyses of ten A549 proteins in replicates using the optimized method yielded fast, reliable characterization, suggesting the potential application of this method in high-throughput protein identification by PMF.     

Identification of proteins from two-dimensional polyacrylamide gels using a novel acid-labile surfactant

Protein identification by peptide mass mapping usually involves digestion of gel-separated proteins with trypsin, followed by mass measurement of the resulting peptides by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Positive identification requires measurement of enough peptide masses to obtain a definitive match with sequence information recorded in protein or DNA sequence databases. However, competitive binding and ionization of residual surfactant introduced during polyacrylamide gel electrophoresis (PAGE) can inhibit solid-phase extraction and MS analysis of tryptic peptides. We have evaluated a novel, acid-labile surfactant (ALS) as an alternative to sodium dodecylsulfate (SDS) for two-dimensional (2-D) PAGE separation and MALDI-MS mapping of proteins. ALS was substituted for SDS at the same concentration in buffers and gels used for 2-D PAGE. Manual and automated procedures for spot cutting and in-gel digestion were used to process Coomassie stained proteins for MS analysis. Results indicate that substituting ALS for SDS during PAGE can significantly increase the number of peptides detected by MALDI-MS, especially for proteins of relatively low abundance. This effect is attributed to decomposition of ALS under acidic conditions during gel staining, destaining, peptide extraction and MS sample preparation. Automated excision and digestion procedures reduce contamination by keratin and other impurities, further enhancing MS identification of gel separated proteins.     

Protein identification and tracking in two-dimensional electrophoretic gels by minimal protein identifiers

Protein identification by matrix-assisted laser desorption/ionization mass-spectrometry peptide mass fingerprinting (MALDI-MS PMF) represents a cornerstone of proteomics. However, it often fails to identify low-molecular-mass proteins, protein fragments, and protein mixtures reliably. To overcome these limitations, PMF can be complemented by tandem mass spectrometry and other search strategies for unambiguous protein identification. The present study explores the advantages of using a MALDI-MS-based approach, designated minimal protein identifier (MPI) approach, for protein identification. This is illustrated for culture supernatant (CSN) proteins of Mycobacterium tuberculosis H37Rv after separation by two-dimensional gel electrophoresis (2-DE). The MPI approach takes into consideration that proteins yield characteristic peptides upon proteolytic cleavage. In this study, peptide mixtures derived from tryptic protein cleavage were analyzed by MALDI-MS and the resulting spectra were compared with template spectra of previously identified counterparts. The MPI approach allowed protein identification by few protein-specific signature peptide masses and revealed truncated variants of mycobacterial elongation factor EF-Tu, previously not identified by PMF. Furthermore, the MPI approach can be employed to track proteins in 2-DE gels, as demonstrated for the 14 kDa antigen, the 10 kDa chaperone, and the conserved hypothetical protein Rv0569 of M. tuberculosis H37Rv. Furthermore, it is shown that the power of the MPI approach strongly depends on distinct factors, most notably on the complexity of the proteome analyzed and accuracy of the mass spectrometer used for peptide mass determination.     

Improving sample treatment for in-solution protein identification by peptide mass fingerprint using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Three ultrasonic energy sources were studied to speed up the sample treatment for in-solution protein identification by peptide mass fingerprint using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Protein reduction, alkylation, and enzymatic digestion steps were done in 15 min. Nine proteins, including zinc resistance-associated protein precursor from Desulfovibrio desulfuricans strain G20 and split-soret cytochrome c from D. desulfuricans ATCC27774 were successfully identified with the new protocol.