Proteomic analysis of cartilage proteins

While the analysis of the cartilage proteome is important for our comprehensive understanding of the development and disease of this important tissue, several unique features of cartilage present some technical obstacles. Firstly, cartilage is difficult to obtain in adequate quantities for many protein analyses, especially from mice which are otherwise powerful experimental models. Furthermore, the cartilage extracellular matrix contains an insoluble network of collagen II-containing fibrils that are integrated within an abundant anionic network of aggrecan and hyaluronan aggregates. These interacting networks provide a structural scaffold for the covalent and non-covalent attachment of other proteins and glycoproteins. Consequently, proteomic analysis of cartilage requires extraction of proteins with chaotropic agents to achieve and significant protein solubilization. Finally, isolated chondrocytes are phenotypically unstable, which requires rapid isolation of cells or the use of specific culture conditions. Despite these problems, recent improvements in the sensitivity and reproducibility of two-dimensional electrophoresis (2-DE) and tandem mass spectrometry (MS/MS) techniques, combined with improved tissue preparation and sample pre-fractionation approaches, have made the proteomic characterization of cartilage tissues possible. Here we review the approaches that have been used and describe in detail protocols for the proteomic analysis of cartilage tissues and cells.     

Investigation of solubilization and digestion methods for microsomal membrane proteome analysis using data-independent LC-MSE

To evaluate the implementation of various denaturants and their efficacy in bottom-up membrane proteomic methods using LC-MS analysis, microsomes isolated from tomato roots were treated with MS-compatible surfactants (RapiGest SF Surfactant from Waters and PPS Silent Surfactant from Protein Discovery), a chaotropic reagent (guanidine hydrochloride), and an organic solvent (methanol). Peptides were analyzed in triplicate sample and technical replicates by data-independent LC-MS(E) analysis. Overall, 2333 unique peptides matching to 662 unique proteins were detected with the order of denaturant method efficacy being RapiGest SF Surfactant, PPS Silent Surfactant, guanidine hydrochloride, and methanol. Using bioinformatic analysis, 103 proteins were determined to be integral membrane proteins. When normalizing the data as a percentage of the overall number of peptides and proteins identified for each method, the order for integral membrane protein identification efficacy was methanol, guanidine hydrochloride, RapiGest SF Surfactant, and PPS Silent Surfactant. Interestingly, only 8% of the proteins were identified in all four methods with the silent surfactants having the greatest overlap at 17%. GRAVY analysis at the protein and peptide level indicated that methanol and guanidine hydrochloride promoted detection of hydrophobic proteins and peptides, respectively; however, trypsin activity in the presence of each denaturant was determined as a major factor contributing to peptide identification by LC-MS(E) . These results reveal the complementary nature of each denaturant method, which can be used in an integrated approach to provide a more effective bottom-up analysis of membrane proteomes than can be achieved using only a single denaturant.     

Comparison of detergent‐based sample preparation workflows for LTQ‐Orbitrap analysis of the Escherichia coli proteome

This work presents a comparative evaluation of several detergent‐based sample preparation workflows for the MS‐based analysis of bacterial proteomes, performed using the model organism Escherichia coli. Initially, RapiGest‐ and SDS‐based buffers were compared for their protein extraction efficiency and quality of the MS data generated. As a result, SDS performed best in terms of total protein yields and overall number of MS identifications, mainly due to a higher efficiency in extracting high molecular weight (MW) and membrane proteins, while RapiGest led to an enrichment in periplasmic and fimbrial proteins. Then, SDS extracts underwent five different MS sample preparation workflows, including: detergent removal by spin columns followed by in‐solution digestion (SC), protein precipitation followed by in‐solution digestion in ammonium bicarbonate or urea buffer, filter‐aided sample preparation (FASP), and 1DE separation followed by in‐gel digestion. On the whole, about 1000 proteins were identified upon LC‐MS/MS analysis of all preparations (>1100 with the SC workflow), with FASP producing more identified peptides and a higher mean sequence coverage. Each protocol exhibited specific behaviors in terms of MW, hydrophobicity, and subcellular localization distribution of the identified proteins; a comparative assessment of the different outputs is presented.     

Enhanced proteomic analysis of Streptomyces peucetius cytosolic protein using optimized protein solubilization protocol

Improvements in the dissolution of proteins in two-dimensional gel electrophoresis have greatly advanced the ability to analyze the proteomes of microorganisms under a wide variety of physiological conditions. This study examined the effect of various combinations of chaotropic agents, a reducing agent, and a detergent on the dissolution of the Streptomyces peucetius cytosolic proteins. The use of urea alone in a rehydration buffer as a chaotropic agent gave the proteome a higher solubility than any of the urea and thiourea combinations, and produced the highest resolution and clearest background in two-dimensional gel electrophoresis. Two % CHAPS, as a detergent in a rehydration buffer, improved the protein solubility. After examining the effect of several concentrations of reducing agent, 50 mM DTT in a rehydration buffer was found to be an optimal condition for the proteome analysis of Streptomyces. Using this optimized buffer condition, more than 2,000 distinct and differentially expressed soluble proteins could be resolved using two-dimensional gel electrophoresis with a pI ranging from 4-7. Under this optimized condition, 15 novel small proteins with low-level expression, which could not be analyzed under the non-optimized conditions, were identified. Overall, the optimized condition helped produce a better reference gel for Streptomyces peucetius.     

Integrated SDS removal and peptide separation by strong-cation exchange liquid chromatography for SDS-assisted shotgun proteome analysis

We report an improved shotgun method for analyzing proteomic samples containing sodium dodecyl sulfate (SDS). This method is based on the use of strong-cation exchange (SCX) liquid chromatography (LC) for SDS removal that can be integrated with peptide separation as the first dimension of the two-dimensional LC tandem mass spectrometry workflow. To optimize the performance of SDS removal, various experimental conditions, including the concentrations of chemical reagents and salts in the sample, the SDS concentration, and the SCX mobile phase composition, were investigated. It was found that a peptide recovery rate of about 90% could be achieved while removing SDS efficiently. One key finding was that, by increasing the SDS concentration to a certain level (0.5%) in the digested peptide sample, the sample recovery rate could be increased. The peptide recovery rate of BSA digests was found to be 90.6 ± 1.0% (n = 3), and SDS in the SCX fractions collected was not detectable by pyrolysis GC-MS, i.e., below the detection limit of 0.00006% for the undesalted SCX fractions. The peptide recovery rates were found to be 90.9% ± 2.7 (n = 3) and 89.5% ± 0.5% (n = 3) for the digests of the membrane-protein-enriched fractions of E. coli cell lysates and the MCF-7 breast cancer cell line, respectively. Compared to the methods that use acid-labile surfactants, such as RapiGest and PPS, for the MCF-7 membrane fraction sample, the SDS method identified, on average (n = 3), more peptides (～5%) and proteins (～16%) than the RapiGest method, while the RapiGest method identified more peptides (～21%) and proteins (～7%) from the E. coli membrane fraction than the SDS method. In both cases, the two methods identified more peptides and proteins than the PPS method. Since SCX is widely used as the first dimension of 2D-LC MS/MS, integration of SDS removal with peptide separation in SCX does not add any extra steps to the sample handling process. We demonstrated the application of this method for 2D-LC MS/MS profiling of the MCF-7 membrane protein fraction and identified 6889 unique peptides, corresponding to 2258 unique proteins or protein groups from two replicate experiments with a false peptide discovery rate of ～0.8%, compared to 5172 unique peptides and 1847 unique proteins identified by the RapiGest method.     

Preparation and application of a partially degradable gel in mass spectrometry-based proteomic analysis

In-gel digestion is an attractive route in mass spectrometry-based proteomic analysis, which, however, often suffers from a certain amount of sample loss mainly due to insufficient protein digestion and peptide extraction. To address this, herein we establish a partially degradable gel-assisted protein digestion and peptide recovery method by means of a simple replacement of bis-acrylamide (BA) with bis-acrylylcystamine (BAC). Concretely, the protein sample solubilized using high concentrations of sodium dodecyl sulfate (SDS) and urea were directly entrapped and immobilized into BAC-crosslinked gel by vacuum-dried gel absorption followed by fixation treatment. After removal of SDS and urea by repeated washing, the proteins were subjected to in-gel digestion and the gel was reductively treated. The tryptic peptides were recovered from the partial degradation of the gel and analyzed afterwards by capillary liquid chromatography coupled with tandem mass spectrometry (CapLC-MS/MS). Compared with conventional BA-crosslinked gel method, this new method increased the numbers of identified proteins and unique peptides by 20.2% and 20.4%, respectively. The further statistical analysis demonstrated that the method improved the recovery of tryptic peptides particularly larger and/or hydrophobic peptides, thereby significantly facilitating protein identification. Thus, the newly developed method is a promising alternative for BA-crosslinked gel-based shotgun workflows and has potential application in the related fields of protein chemistry and proteomics.     

厌氧产氢颗粒污泥蛋白质组分析样品的高效制备方法

【背景】厌氧产氢颗粒污泥比絮状产氢污泥具有更高的生物量、沉降性与反应效率,对颗粒污泥进行蛋白质组学研究,有助于揭示其代谢调控的分子机制,从而对厌氧代谢过程进行优化调控。目前关于产氢颗粒污泥蛋白质组分析样品制备方法的研究尚未见文献报道。革兰氏阳性菌Ethanoligenens harbinense YUAN-3是自凝集产氢发酵细菌,在间歇和连续流培养中可形成自聚集的厌氧颗粒,由于其全基因组信息清楚,可作为模式研究材料对制备方法进行评估。【目的】针对厌氧产氢颗粒污泥的蛋白质组学研究,比较不同蛋白质提取方法进行优化。【方法】分别利用液氮研磨、超声破碎、匀浆破碎对产氢颗粒污泥破碎,比较这3种方法对总蛋白提取量的影响;通过双向电泳比较三氯乙酸(Trichloroacetic acid,TCA)-丙酮沉淀法与苯酚抽提法对总蛋白提取效果的影响;对总蛋白样品分别进行同位素标记相对和绝对定量标记(Isobarictagsforrelativeandabsolutequantification,i TRAQ)、串联质谱标签(Tandemmasstag,TMT)标记以及质谱鉴定。【结果】液氮研磨、超声破碎、匀浆破碎3种破碎方法下总蛋白的提取量分别是对照样品的2.0、3.9与5.2倍。与TCA-丙酮沉淀法相比,苯酚抽提法总蛋白样品在双向电泳图谱上的蛋白质点明显增多,分布均匀,同时其在碱性蛋白端与小分子量蛋白端的蛋白质点也明显增多。质谱分析发现,iTRAQ标记样品与TMT标记样品中分别鉴定到1797个与1644个蛋白,在分子量、等电点、亚细胞定位的各个分布范围内,这些蛋白良好地覆盖了E.harbinenseYUAN-3中各个类型的蛋白。【结论】匀浆破碎与苯酚抽提法联用的总蛋白制备方法更适用于厌氧产氢颗粒污泥,该方法有利于后续的蛋白质双向电泳和定量蛋白质组质谱分析,可作为产氢颗粒污泥以及革兰氏阳性菌总蛋白制备的方法参考。     

Sodium-deoxycholate-assisted tryptic digestion and identification of proteolytically resistant proteins

Identification of proteolytically resistant proteins with compact molecular structure and/or poor water solubility is a challenge in current proteomic study. In this study, sodium deoxycholate (SDC)-assisted tryptic digestion and identification of proteolytically resistant myoglobin and integral membrane proteins were systematically investigated. When the effect of SDC up to 10% on trypsin activity was investigated, little decrease in the trypsin activity was observed in 1% SDC solution, 2-5% SDC decreased the enzyme activity only by about 13.6%, and even in the presence of 10% SDC trypsin still retained 77.4% of its activity. Matrix-assisted laser desorption ionization time of flight mass spectrometry analysis showed that SDC could be removed from sample solution with acid treatment followed by centrifugation, and the remaining SDC, if any, had little effect on mass spectrometry analysis with regard to the number and signal/noise ratio of ions in the mass spectra. Compared with urea and methanol, two other commonly used additives in addition to SDS in proteomic analysis, SDC improved more efficiently the denaturation, solubilization, and tryptic digestion of proteins, particularly proteolytically resistant myoglobin and integral membrane proteins, thereby enhancing the efficiency of their identification with regard to the number of identified proteins and unique peptides and the sequence coverage of matched proteins.     

A method for the isolation of covalent DNA-protein crosslinks suitable for proteomics analysis

The covalent crosslinking of protein to DNA is a form of DNA damage induced by a number of commonly encountered agents, including metals, aldehydes, and radiation as well as chemotherapeutic drugs. DNA-protein crosslinks (DPCs) are potentially bulky and helix distorting and have the potential to block the progression of translocating protein complexes. To fully understand the induction and repair of these lesions, it will be important to identify the crosslinked proteins involved. To take advantage of dramatic improvements in instrument sensitivity that have facilitated the identification of proteins by proteomic approaches, improved methods are required for isolation of DPCs. This article describes a novel method for the isolation of DPCs from mammalian cells that uses chaotropic agents to isolate genomic DNA and stringently remove noncrosslinked proteins followed by DNase I digestion to release covalently crosslinked proteins. This method generates high-quality protein samples in sufficient quantities for analysis by mass spectrometry. In addition, the article presents a modified form of this method that also makes use of chaotropic agents for promoting the adsorption of DNA (with crosslinked proteins) to silica fines, markedly reducing the DPC isolation time and cost. These approaches were applied to radiation- and camptothecin-induced DPCs.     

Evaluation of the combination of bead technology with SELDI-TOF-MS and 2-D DIGE for detection of plasma proteins

Today biomarker discovery is one of the most active aspects of proteomic investigations. However, the wide dynamic range of plasma proteins makes the analysis very challenging because high abundance proteins tend to mask those of lower abundance. Using a large bead-based library of combinatorial peptide ligands (Equalizer beads or ProteoMiner), the dynamic range of the protein concentration is compressed, the high abundance proteins present in the sample are reduced and the low abundance proteins are enriched, while retaining representatives of all proteins within the sample. In the present study, the combination of beads with surface enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) and two-dimensional differential gel electrophoresis (2-D DIGE) technology were evaluated considering efficiency, reproducibility, sensitivity, and compatibility. The bead technology is easily compatible with both SELDI-TOF-MS and 2-D DIGE and the samples can be analyzed directly without any processing of the sample. The use of the beads prior SELDI-TOF-MS and 2-D DIGE enabled detection of many new protein spots/peaks and increased resolution and improved intensity of low abundance proteins in a reproducible fashion compared with the depletion technique. Several proteins have been identified by the combination of beads, 2-D DIGE and MS for example different kinds of complement factors and cytoskeletal proteins. Our data suggest that integration of the bead technology with our current proteomic technologies will enhance the possibility to deliver new peptide/protein biomarker candidates in our projects.     

Osteogenic differentiation of adipose derived stem cells promoted by overexpression of osterix

Secreted proteins, which may be involved in the regulation of various biological processes, are the potential targets for diagnosis and treatment of diverse diseases. In this study, to identify the human hepatoma HepG2 cells-derived secreted proteins more extensively, we applied the protein sample preparations using the combinations of denaturation methods and molecular-mass cutoff via ultrafiltration to the two-dimensional liquid chromatography coupled with tandem mass spectrometry (2D LC–MS/MS) analysis. We were able to identify a total of 86 proteins containing widely known secreted proteins of HepG2 such as alpha-fetoprotein, of which 73 proteins including 27 signal peptide-containing proteins have never been reported to be secreted from HepG2 cells in other proteomic studies. Among the identified signal peptide-containing proteins, ten proteins such as growth differentiation factor 15, osteopontin and stanniocalcin 2 were discovered as new secreted proteins of HepG2 cells. These observations suggest that the combinations of different sample preparation methods and 2D LC–MS/MS analysis are useful for identifying a wider range of low-abundance proteins and that the secreted proteins from HepG2 identified in this study may be useful as liver-specific biomarkers for diagnosis and treatment.     

A catalogue of human saliva proteins identified by free flow electrophoresis-based peptide separation and tandem mass spectrometry

Human saliva has great potential for clinical disease diagnostics. Constructing a comprehensive catalogue of saliva proteins using proteomic approaches is a necessary first step to identifying potential protein biomarkers of disease. However, because of the challenge presented in cataloguing saliva proteins with widely varying abundance, new proteomic approaches are needed. To this end, we used a newly developed approach coupling peptide separation using free flow electrophoresis with linear ion trap tandem mass spectrometry to identify proteins in whole human saliva. We identified 437 proteins with high confidence (false positive rate below 1%), producing the largest catalogue of proteins from a single saliva sample to date and providing new information on the composition and potential diagnostic utility of this fluid. The statistically validated, transparently presented, and annotated dataset provides a model for presenting large scale proteomic data of this type, which should facilitate better dissemination and easier comparisons of proteomic datasets from future studies in saliva.     

Inhibition of protein carbamylation in urea solution using ammonium-containing buffers

Urea solution is one of the most commonly employed protein denaturants for protease digestion in proteomic studies. However, it has long been recognized that urea solution can cause carbamylation at the N termini of proteins/peptides and at the side chain amino groups of lysine and arginine residues. Protein/peptide carbamylation blocks protease digestion and affects protein identification and quantification in mass spectrometry analysis by blocking peptide amino groups from isotopic/isobaric labeling and changing peptide charge states, retention times, and masses. In addition, protein carbamylation during sample preparation makes it difficult to study in vivo protein carbamylation. In this study, we compared the peptide carbamylation in urea solutions of different buffers and found that ammonium-containing buffers were the most effective buffers to inhibit protein carbamylation in urea solution. The possible mechanism of carbamylation inhibition by ammonium-containing buffers is discussed, and a revised procedure for the protease digestion of proteins in urea and ammonium-containing buffers was developed to facilitate its application in proteomic research.     

Exploitation of specific properties of trifluoroethanol for extraction and separation of membrane proteins

Hydrophobic proteins are difficult to analyze by two-dimensional electrophoresis (2-DE) because of their intrinsic tendency to self-aggregate during the first dimension (isoelectric focusing, IEF) or the equilibration steps. This aggregation renders their redissolution for the second dimension uncertain and results in the reduction of the number and intensity of protein spots, and in undesirable vertical and horizontal streaks across gels. Trifluoroethanol (TFE) is traditionally used at high concentration to solubilize peptides and proteins for NMR studies. Depending upon its concentration, TFE strongly affects the three-dimensional structure of proteins. We report here a phase separation system based on TFE/CHCl(3), which is able to extract a number of intrinsic membrane proteins. The addition of TFE in the in-gel sample rehydration buffer to improve membrane protein IEF separation is also presented. The procedure using urea, thiourea, and sulfobetaine as chaotropic agents was modified by the addition of TFE and removing of sulfobetaine at an optimized concentration in the solubilization medium used for the first dimension. When using membrane fractions isolated from Escherichia coli, the intensity and the number of spots detected from 2-DE gels that used TFE in the solubilization medium were significantly increased. The majority of the proteins identified using peptide mass fingerprinting and tandem mass spectrometry (MS/MS) were intrinsic membrane proteins, proteins of beta barrel structure or transmembrane proteins.     

Large-scale proteomic analysis of membrane proteins

Proteomic analysis of membrane proteins is a promising approach for the identification of novel drug targets and/or disease biomarkers. Despite notable technological developments, obstacles related to extraction and solublization of membrane proteins are encountered. A critical discussion of the different preparative methods of membrane proteins is offered in relation to downstream proteomic applications, mainly gel-based analyses and mass spectrometry. Frequently, unknown proteins are identified by high-throughput profiling of membrane proteins. In search for novel membrane proteins, analysis of protein sequences using computational tools is performed to predict the presence of transmembrane domains. This review also presents these bioinformatic tools with the human proteome as a case study. Along with technological innovations, advancements in the areas of sample preparation and computational prediction of membrane proteins will lead to exciting discoveries.     

Large-scale proteomic analysis of membrane proteins

Proteomic analysis of membrane proteins is a promising approach for the identification of novel drug targets and/or disease biomarkers. Despite notable technological developments, obstacles related to extraction and solublization of membrane proteins are encountered. A critical discussion of the different preparative methods of membrane proteins is offered in relation to downstream proteomic applications, mainly gel-based analyses and mass spectrometry. Frequently, unknown proteins are identified by high-throughput profiling of membrane proteins. In search for novel membrane proteins, analysis of protein sequences using computational tools is performed to predict the presence of transmembrane domains. This review also presents these bioinformatic tools with the human proteome as a case study. Along with technological innovations, advancements in the areas of sample preparation and computational prediction of membrane proteins will lead to exciting discoveries.     

Comparison of protein solubilization methods suitable for proteomic analysis of soybean seed proteins

Extraction of soybean seed proteins for two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry analysis is challenging and inconsistent. In this study, we compared four different protein extraction/solubilization methods-urea, thiourea/urea, phenol, and a modified trichloroacetic acid (TCA)/acetone-to determine their efficacy in separating soybean seed proteins by 2D-PAGE. In all four methods, seed storage proteins were well separated by 2D-PAGE with minor variations in the intensity of the spots. The thiourea/urea and TCA methods showed higher protein resolution and spot intensity of all proteins compared with the other two methods. In addition, several less abundant and high molecular weight proteins were clearly resolved and strongly detected using the thiourea/urea and TCA methods. Protein spots obtained from the TCA method were subjected to mass spectrometry analysis to test their quality and compatibility. Fifteen protein spots were selected, digested with trypsin, and analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and liquid chromatography mass spectrometry (LC-MS). The proteins identified were beta-conglycinin, glycinin, Kunitz trypsin inhibitor, alcohol dehydrogenase, Gly m Bd 28K allergen, and sucrose binding proteins. These results suggest that the thiourea/urea and TCA methods are efficient and reliable methods for 2D separation of soybean seed proteins and subsequent identification by mass spectrometry.     

Proteomic analysis of formalin-fixed paraffin-embedded tissue by MALDI imaging mass spectrometry

Archived formalin-fixed paraffin-embedded (FFPE) tissue collections represent a valuable informational resource for proteomic studies. Multiple FFPE core biopsies can be assembled in a single block to form tissue microarrays (TMAs). We describe a protocol for analyzing protein in FFPE-TMAs using matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS). The workflow incorporates an antigen retrieval step following deparaffinization, in situ trypsin digestion, matrix application and then mass spectrometry signal acquisition. The direct analysis of FFPE-TMA tissue using IMS allows direct analysis of multiple tissue samples in a single experiment without extraction and purification of proteins. The advantages of high speed and throughput, easy sample handling and excellent reproducibility make this technology a favorable approach for the proteomic analysis of clinical research cohorts with large sample numbers. For example, TMA analysis of 300 FFPE cores would typically require 6 h of total time through data acquisition, not including data analysis.     

类芽孢杆菌(Paenibacillus sp.)蛋白质组学研究中三种蛋白提取方法的比较分析

【目的】革兰氏阳性类芽孢杆菌（Paenibacillus sp.）本身细胞壁的结构特点导致其菌体全蛋白不易获得。本研究选取了3种破碎方法——溶菌酶联合超声破碎法（方法一）、溶菌酶联合SDS热处理破碎法（方法二）、液氮联合超声破碎法（方法三）进行革兰氏阳性菌的细胞破碎,以期获得适于样品菌株基于质谱技术进行蛋白质组学研究的制备方法。【方法】在蛋白样品的制备过程中,对3种不同破碎方法的蛋白提取得率和SDS-PAGE检测分析结果进行比较;随后将3种蛋白样品制备方法的样品用质谱技术进行鉴定,分析不同蛋白样品基于质谱技术鉴定蛋白的差异。【结果】在蛋白样品的制备提取过程中,不同破碎方法的蛋白提取率大致相同。用单因素方差比较3种提取方法质谱鉴定蛋白数的差异性,方法三鉴定的蛋白数最多（2 638个）,其次是方法一（2 452个）,方法二鉴定的蛋白数最少（2 003个）。进一步用韦恩图分析比较不同提取方法的蛋白鉴定通量差异,综合考虑蛋白提取效率的结果以及液氮研磨法提取蛋白的缺点,最终选取溶菌酶联合超声破碎法（方法一）提取菌株全蛋白作为该菌基于质谱分析其蛋白质组学研究中最适合的方法。最后,对质谱鉴定菌株蛋白包括分子量、等电点、疏水性的基本性质进行分析,发现3种破碎方法质谱鉴定的蛋白与模式菌株多黏类芽孢杆菌（Paenibacillus polymyxa）基因组中预测蛋白的各个组分分布占比基本一致,都保证了菌株蛋白质组数据信息的完整性。【结论】基于质谱技术开展革兰氏阳性类芽孢杆菌（Paenibacillus sp.）的蛋白质组学研究,溶菌酶联合超声破碎法是提取该菌株全蛋白最适合的方法。