Separation with zwitterionic hydrophilic interaction liquid chromatography improves protein identification by matrix‐assisted laser desorption/ionization‐based proteomic analysis

Comprehensive proteomic analyses necessitate efficient separation of peptide mixtures for the subsequent identification of proteins by mass spectrometry (MS). However, digestion of proteins extracted from cells and tissues often yields complex peptide mixtures that confound direct comprehensive MS analysis. This study investigated a zwitterionic hydrophilic interaction liquid chromatography (ZIC‐HILIC) technique for the peptide separation step, which was verified by subsequent MS analysis. Human serum albumin (HSA) was the model protein used for this analysis. HSA was digested with trypsin and resolved by ZIC‐HILIC or conventional strong cation exchange (SCX) prior to MS analysis for peptide identification. Separation with ZIC‐HILIC significantly improved the identification of HSA peptides over SCX chromatography. Detailed analyses of the identified peptides revealed that the ZIC‐HILIC has better peptide fractionation ability. We further demonstrated that ZIC‐HILIC is useful for quantitatively surveying cell surface markers specifically expressed in undifferentiated embryonic stem cells. These results suggested the value of ZIC‐HILIC as a novel and efficient separation method for comprehensive and quantitative proteomic analyses. Copyright © 2009 John Wiley & Sons, Ltd.     

原子转移自由基聚合修饰银丝固定化酶反应器的制备及在蛋白质组鉴定中的应用

针对传统溶液酶解存在的酶解时间较长、酶自切物干扰以及蛋白酶不能重复使用等缺陷,通过电子转移生成催化剂的原子转移自由基聚合法修饰银丝,并以其为载体制备了一种新型的固定化酶反应器。用质谱考察了银丝固定化酶反应器(SW-Trypsin)的酶解效率、重复性和回收率。结果表明:绒毛状聚合物修饰的SW-Trypsin的酶解效率较高,酶解标准蛋白牛血清白蛋白(BSA)20 min后,肽段的氨基酸序列覆盖率可达93%,高于传统溶液酶解方法酶解16 h所得79%的覆盖率。使用该固定化酶反应器于一个月内8次酶解BSA所得的氨基酸序列覆盖率在89%到97%之间,平均覆盖率为94%,显示出良好的稳定性。另外,该固定化酶反应器酶解马心肌红蛋白(MYO)的回收率为87.67%。最后,用SW-Trypsin酶解腾冲嗜热菌全蛋白20 min,所鉴定到的氨基酸序列覆盖率和蛋白数量与同样条件下溶液酶解16 h的结果接近,且零漏切位点肽段的比例更高。加之容易分离的优点,SW-Trypsin在蛋白质组学的应用中具有良好的前景。     

On-column digestion of proteins in aqueous-organic solvents

Proteolytic digestion is an important step in protein identification by peptide mass mapping and tandem mass spectrometry (MS/MS)-based peptide sequencing. Traditional methods of protein digestion require extended incubation times and have difficulty with proteolytically resistant proteins. Here, we describe a method in which a protein solution was combined with a mixed aqueous-organic solution (methanol, isopropanol, or acetonitrile) and passed through a microcolumn containing immobilized trypsin. Myoglobin sequence coverage was high (>85%) in all three solvents, and differences in spectra were seen among the different solution conditions. Notably, methanol-based digestions produced fewer missed cleavages while acetonitrile-based digestions produced the most peptides and the most intense mass spectra. Flow rates through the column were varied from 0.5 to 15 micro L/min, corresponding to column residence times of 78 and 2.6 s, respectively. All flow rates produced high sequence coverage of myoglobin, although, at higher flow rates, more missed cleavages were observed. No significant increase in undigested myoglobin was observed with flow rates up to 15 micro L/min. The described method was applied to the digestion of human transferrin (hTf), a proteolytically resistant protein. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOFMS) analysis detected 42 peptides covering 46% of the hTf sequence. The traditional aqueous method resulted in 12 peptides (8% sequence coverage) only when high concentrations of trypsin were used. Lastly, digestion of low nanomolar myoglobin was shown to produce detectable peptides and resulted in a correct database hit. Thus, we demonstrate a method that is capable of rapid on-line digestion, thereby lending itself to high-throughput identification of proteins.     

Disparities between immobilized enzyme and solution based digestion of transferrin with trypsin

Trypsin digestion is a major component of preparing proteins for peptide based identification and quantification by mass spectral (MS) analysis. Surprisingly proteolysis is the slowest part of the proteomics process by an order of magnitude. Numerous recent efforts to reduce protein digestion to a few minutes have centered on the use of an immobilized enzyme reactor (IMER) to minimize both trypsin autolysis and vastly increase the trypsin to protein ratio. A central question in this approach is whether proteolysis with an IMER produces the same peptide cleavage products as derived from solution based digestion. The studies reported here examined this question with transferrin; a model protein of known resistance to trypsin digestion. Results from these studies confirmed that a trypsin‐IMER can in fact digest transferrin in a few minutes; providing tryptic peptides that subsequent to MS analysis allow sequence identification equivalent to solution digestion. Although many of the peptides obtained from these two trypsin digestion systems were identical, many were not. The greatest difference was that the trypsin‐ IMER produces (i) numerous peptides bearing multiple lysine and/or arginine residues and (ii) identical portions of the protein sequence were found in multiple peptides. Most of these peptides were derived from five regions in transferrin. These results were interpreted to mean that proteolysis in the case of transferrin occurred faster than the rate at which buried lysine and arginine residues were unmasked in the five regions providing peptides that were only partially digested.     

Use of monolithic supports in proteomics technology

An overview on the utilization of monoliths in proteomics technology will be given. Both silica- and polymer-based monoliths have broad use for microseparation of tryptic peptides in reversed-phase (RP) mode before identification by mass spectrometry (MS) or by MS/MS. For two-dimensional (2D) LC separation of peptides before MS or MS/MS analysis, a combination of ion-exchange, usually cation-exchange (CEX) chromatography with RP chromatography on monolithic supports can be employed. Immobilized metal ion affinity chromatography monoliths with immobilized Fe3+-ions are used for the isolation of phosphopeptides. Monoliths with immobilized affinity ligands are usually applied to the rapid separation of proteins and peptides. Miniaturized reactors with immobilized proteolytic enzymes are utilized for rapid on- or offline digestion of isolated proteins or protein mixtures prior to identification by LC-MS/MS. Monoliths also have broad potential for application in sample preparation, prior to further proteomic analyses. Monolithic supports with large pore sizes can be exploited for the isolation of nanoparticles, such as cells, organelles, viruses and protein aggregates. The potential for further adoption of monolithic supports in protein separation and enrichment of low abundance proteins prior to proteolytic digestion and final LC-MS/MS protein identification will be discussed.     

High-speed biomarker identification utilizing porous silicon nanovial arrays and MALDI-TOF mass spectrometry

Speed and accuracy are crucial prerequisites in the application of proteomic methods to clinical medicine. We describe a microfluidic-based nanovial array for rapid proteolytic processing linked to MALDI-TOF MS. This microscale format consumes only minute amounts of sample, and it is compatible with rapid bioanalytical protocols and high-sensitivity readouts. Arrays of vials (300 microm in diameter and 25 microm deep), isotropically etched in silicon wafers were electrochemically porosified. Automated picoliter microdispensing was employed for precise fluid handling in the microarray format. Vials were prefilled with trypsin solution, which was allowed to dry. Porosified and nonporosified nanovials were compared for trypsin digestion and subsequent MS identification of three model proteins: lysozyme, alcohol dehydrogenase, and serum albumin at levels of 100 and 20 fmol. In an effort to assess the rapid digestion platform in a context of putative clinical applications, two prostate cancer biomarkers, prostate-specific antigen (PSA) and human glandular kallikrein 2 (hK2), were digested at levels of 100 fmol (PSA), 20 fmol (PSA) and 8 fmol (hK2). All biomarker digestions were completed in less than 30 s, with successful MS identification in the porous nanovial setting.     

Improvement of gel‐separated protein identification by DMF‐assisted digestion and peptide recovery after electroblotting

In‐gel digestion of gel‐separated proteins is a major route to assist in proteomics‐based biological discovery, which, however, is often embarrassed by its inherent limitations such as the low digestion efficiency and the low recovery of proteolytic peptides. For overcoming these limitations, many efforts have been directed at developing alternative methods to avoid the in‐digestion. Here, we present a new method for efficient protein digestion and tryptic peptide recovery, which involved electroblotting gel‐separated proteins onto a PVDF membrane, excising the PVDF bands containing protein of interest, and dissolving the bands with pure DMF (≥99.8%). Before tryptic digestion, NH₄HCO₃ buffer was added to moderately adjust the DMF concentration (to 40%) in order for trypsin to exert its activity. Experimental results using protein standards showed that, due to actions of DMF in dissolving PVDF membrane and the membrane‐bound substances, the proteins were virtually in‐solution digested in DMF‐containing buffer. This protocol allowed more efficient digestion and peptide recovery, thereby increasing the sequence coverage and the confidence of protein identification. The comparative study using rat hippocampal membrane‐enriched sample showed that the method was superior to the reported on‐membrane tryptic digestion for further protein identification, including low abundant and/or highly hydrophobic membrane proteins.     

An ultra-fast and highly efficient multiple proteases digestion strategy using graphene-oxide-based immobilized protease reagents

Highly efficient and rapid proteolytic digestion of proteins into peptides is a crucial step in shotgun-based proteome-analysis strategy.Tandem digestion by two or more proteases is demonstrated to be helpful for increasing digestion efficiency and decreasing missed cleavages,which results in more peptides that are compatible with mass-spectrometry analysis.Compared to conventional solution digestion,immobilized protease digestion has the obvious advantages of short digestion time,no self-proteolysis,and reusability.We proposed a multiple-immobilized proteases-digestion strategy that combines the advantages of the two digestion strategies mentioned above.Graphene-oxide(GO)-based immobilized trypsin and endoproteinase Glu-C were prepared by covalently attaching them onto the GO surface.The prepared GO-trypsin and GO-Glu-C were successfully applied in standard protein digestion and multiple immobilized proteases digestion of total proteins of Thermoanaerobacter tengcongensis.Compared to 12-hour solution digestion using trypsin or Glu-C,14%and 7%improvement were obtained,respectively,in the sequence coverage of BSA by one-minute digestion using GO-trypsin and GO-Glu-C.Multiple immobilized-proteases digestion of the total proteins of Thermoanaerobacter tengcongensis showed 24.3%and 48.7%enhancement in the numbers of identified proteins than was obtained using GO-trypsin or GO-Glu-C alone.The ultra-fast and highly efficient digestion can be contributed to the high loading capacity of protease on GO,which leads to fewer missed cleavages and more complete digestion.As a result,improved protein identification and sequence coverage can be expected.     

Sequencing Lys-N Proteolytic Peptides by ESI and MALDI Tandem Mass Spectrometry

In this study, we explored the MS/MS behavior of various synthetic peptides that possess a lysine residue at the N-terminal position. These peptides were designed to mimic peptides produced upon proteolysis by the Lys-N enzyme, a metalloendopeptidase issued from a Japanese fungus Grifola frondosa that was recently investigated in proteomic studies as an alternative to trypsin digestion, as a specific cleavage at the amide X-Lys chain is obtained that provides N-terminal lysine peptide fragments. In contrast to tryptic peptides exhibiting a lysine or arginine residue solely at the C-terminal position, and are thus devoid of such basic amino acids within the sequence, these Lys-N proteolytic peptides can contain the highly basic arginine residue anywhere within the peptide chain. The fragmentation patterns of such sequences with the ESI-QqTOF and MALDI-TOF/TOF mass spectrometers commonly used in proteomic bottom-up experiments were investigated.     

Novel staining-free proteomic method for simultaneous identification of proteins and determination of their pI values by using low-molecular-mass pI markers

A new proteomic staining-free method for simultaneous identification of proteins and determination of their pI values by using low-molecular-mass pI markers is described. It is based on separation of proteins in gels by IEF in combination with mass spectrometric analysis of both peptides derived by in-gel digestion and low-molecular-mass pI markers extracted form the same piece excised from the gel. In this method, the pI markers are mixed with a protein mixture (a commercial malted barley protein extract) deposited on a gel and separated in a pH gradient. Color pI markers enable supervision of progress of focusing process. Several separated bands of the pI markers (including separated proteins) were excised and the pI markers were eluted from each gel piece by water/ethanol and identified by MALDI-TOF/TOF MS. The remaining carrier ampholytes were then washed out from gel pieces and proteins were in-gel digested with trypsin or chymotrypsin. Obtained peptides were measured by MALDI-TOF/TOF MS and proteins were identified via protein database search. This procedure allows omitting time-consuming protein staining and destaining procedures, which shortens the analysis time. For comparison, other IEF gels were stained with CBB R 250 and proteins in the gel bands were identified. Similarity of the results confirmed that our approach can give information about the correct pI values of particular proteins in complex samples at significantly shorter analysis times. This method can be very useful for identification of proteins and their post-translational modifications in prefractioned samples, where post-translational modifications (e.g., glycation) are frequent.     

新型鳞片状聚合物修饰硅胶填料固定化酶的制备及其在蛋白质组鉴定中的应用

利用原子转移自由基聚合法制备了鳞片状聚合物修饰的硅胶填料,将其作为一种新型的固定化酶载体,实现了蛋白酶的高密度固定,从而明显缩短了复杂蛋白质样品的酶解时间。使用标准蛋白质对固定化酶的酶解效率进行了考察,结果表明: 鳞片状聚合物修饰的新型固定化酶硅胶填料具有较高的酶解效率,酶解标准蛋白质1 min后,鉴定到肽段的氨基酸序列覆盖率可达95%以上。将该固定化酶硅胶填料成功应用于大肠杆菌全蛋白质的酶解,从2 min酶解肽段的混合物中鉴定到的蛋白质数量超过同样条件下溶液酶解12 h的结果。另外,该固定化酶硅胶填料可以重复使用,其酶解效率具有良好的稳定性和重现性;该固定化酶具有较好的样品回收率,因而可以应用于蛋白质组学研究中。     

A microchip-based proteolytic digestion system driven by electroosmotic pumping

Microchip-based proteomic analysis requires proteolytic digestion of proteins in microdevices. Enzyme reactors in microdevices, fabricated in glass, silicon, and PDMS substrates, have recently been demonstrated for model protein digestions. The common approach used for these enzyme reactors is employment of a syringe pump(s) to generate hydrodynamic flow, driving the proteins through the reactors. Here we present a novel approach, using electroosmotic flow (EOF) to electrokinetically pump proteins through a proteolytic system. The existence of EOF in the proteolytic system packed with immobilized trypsin gel beads was proven by imaging the movement of a neutral fluorescent marker. Digestions of proteins were subsequently carried out for 12 min, and the tryptic peptides were analyzed independently using capillary electrophoresis (CE) and MALDI-TOF mass spectrometry (MS). The results from CE analysis of the tryptic peptides from the EOF-driven proteolytic system and a conventional water bath digestion were comparable. MALDI-TOF MS was used to identify the parent protein and the tryptic peptides using MS-Fit database searching. The potential utility of the EOF-driven proteolytic system was demonstrated by direct electro-elution of proteins from an acrylamide gel into the proteolytic system, with elution and tryptic digestion achieved in a single step. The EOF-driven proteolytic system, thus, provides a simple way to integrate protein digestion into an electrophoretic micro total analysis system for protein analysis and characterization.     

Determination of the glycation sites of Bacillus anthracis neoglycoconjugate vaccine by MALDI-TOF/TOF-CID-MS/MS and LC-ESI-QqTOF-tandem mass spectrometry

We present herein an efficient mass spectrometric method for the localization of the glycation sites of a model neoglycoconjugate vaccine formed by a construct of the tetrasaccharide side chain of the Bacillus anthracis exosporium and the protein carrier bovine serum albumin. The glycoconjugate was digested with both trypsin and GluC V8 endoproteinases, and the digests were then analyzed by MALDI-TOF/TOF-CID-MS/MS and nano-LC-ESI-QqTOF-CID-MS/MS. The sequences of the unknown peptides analyzed by MALDI-TOF/TOF-CID-MS/MS, following digestion with the GluC V8 endoproteinase, allowed us to recognize three glycopeptides whose glycation occupancies were, respectively, on Lys 235, Lys 420, and Lys 498. Similarly, the same analysis was performed on the tryptic digests, which permitted us to recognize two glycation sites on Lys 100 and Lys 374. In addition, we have also used LC-ESI-QqTOF-CID-MS/MS analysis for the identification of the tryptic digests. However, this analysis identified a higher number of glycopeptides than would be expected from a glycoconjugate composed of a carbohydrate-protein ratio of 5.4:1, which would have resulted in glycation occupancies of 18 specific sites. This discrepancy was due to the large number of glycoforms formed during the synthetic carbohydrate-spacer-carrier protein conjugation. Likewise, the LC-ESI-QqTOF-MS/MS analysis of the GluC V8 digest also identified 17 different glycation sites on the synthetic glycoconjugate.     

Mass spectrometric analysis of enzymatic digestion of denatured collagen for identification of collagen type

Collagen type II and I from bovine were thermally denatured and digested with trypsin. The digest mixture was analyzed with liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS). Peptides in the digest mixture were identified by mass spectrometry/mass spectrometry (MS/MS) sequencing. The results indicated that the digest mixtures of collagen type II and I contained lots of specific peptides and common peptides. Specific peptides could be used as index for identifying collagen type. Articular cartilage from bovine was pretreated and analyzed with the same method to determine the collagen types. The result indicated that the method developed was effective for identification of collagen types. The research provided a possible approach for collagen identification in particular tissues.     

Acid-labile surfactant improves in-sodium dodecyl sulfate polyacrylamide gel protein digestion for matrix-assisted laser desorption/ionization mass spectrometric peptide mapping

Mass spectrometry (MS) together with genome database searches serves as a powerful tool for the identification of proteins. In proteome analysis, mixtures of cellular proteins are usually separated by sodium dodecyl sulfate (SDS) polyacrylamide gel-based two-dimensional gel electrophoresis (2-DE) or one-dimensional gel electrophoresis (1-DE), and in-gel digested by a specific protease. In-gel protein digestion is one of the critical steps for sensitive protein identification by these procedures. Efficient protein digestion is required for obtaining peptide peaks necessary for protein identification by MS. This paper reports a remarkable improvement of protein digestion in SDS polyacrylamide gels using an acid-labile surfactant, sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate (ALS). Pretreatment of gel pieces containing protein spots separated by 2-DE with a small amount of ALS prior to trypsin digestion led to increases in the digested peptides eluted from the gels. Consistently, treatment of gel pieces containing silver-stained standard proteins and those separated from tissue extracts resulted in the detection of increased numbers of peptide peaks in spectra obtained by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOFMS). Hence the present protocol with ALS provides a useful strategy for sensitive protein identification by MS.     

Protein proteolysis and the multi-dimensional electrochromatographic separation of histidine-containing peptide fragments on a chip

This paper reports a system for three-dimensional electrochromatography in a chip format. The steps involved included trypsin digestion, copper(II)-immobilized metal affinity chromatography [Cu(II)-IMAC] selection of histidine-containing peptides, and reversed-phase capillary electrochromatography of the selected peptides. Trypsin digestion and affinity chromatography were achieved in particle-based columns with a microfabricated frit whereas reversed-phase separations were executed on a column of collocated monolithic support structures. Column frits were designed to maintain constant cross sectional area and path length in all channels and to retain particles down to a size of 3 microm. Cu(II)-IMAC selection of histidine-containing peptides from standard peptide mixtures and protein digests followed by reversed-phase chromatography of the selected peptides was demonstrated in the electrochromatography mode. The possibility to run a comprehensive proteomic analysis by combining trypsin digestion, affinity selection, and a reversed-phase separation on chips was shown using fluorescein isothiocyanate-labeled bovine serum albumin as an example.     

基于镜像酶正交酶切的蛋白质复合物规模化精准分析新方法

蛋白质主要以复合物的形式参与各项生命活动.化学交联质谱(CXMS)技术作为近年来新兴的蛋白质复合物解析技术,不仅可实现蛋白质复合物规模化解析,而且普遍适用于任意相对分子质量和纯度的蛋白质复合物样品,因此已成为X-射线晶体衍射技术、冷冻电镜技术等蛋白质复合物解析经典技术的重要补充.目前,CXMS主要采用胰蛋白酶将交联后的...     

An integrated workflow for extraction and solubilization of intermediate filaments from colorectal biopsies for proteomic analysis

We report a technique for isolation and solubilization of intermediate filament (IF) proteins from colonic biopsies compatible with both gel electrophoresis and liquid chromatography "shotgun" proteomics using mass spectrometry (MS). This is important because changes in the IF proteome, particularly in keratin expression and modification, are noted in colonic mucosa of patients with colorectal cancer. Though keratins have traditionally been dissolved in high concentration of urea, the latter solvent precludes efficient proteolytic digestion by trypsin prior to gel-free LC-MS/MS approaches. The extraction of cytoskeletal proteins was initially evaluated using MCF-7 cancer cell lines using a published, differential detergent solubilization protocol. IF proteins were extracted from colonic biopsies using a combination of homogenization and sonication. Since comparable efficiency of solubilization was noted on the extracted IF from cell lines between urea and guanidine hydrochloride (GuHCl) in triethylammonium bicarbonate buffer, isolated proteins from endoscopic biopsies were solubilized in GuHCl. Using immunoblotting techniques, we successfully demonstrated isolation of keratins and preservation of posttranslational modifications (phosphorylation, acetylation). Dissolved proteins were tryptically digested and peptides analyzed by MS, showing the functionality of the workflow in shotgun proteomic applications, specifically compatibility of the workflow for isobaric tagging relative and absolute quantification based quantitation approaches.     

反相液相色谱-串联质谱法鉴定油菜蜂花粉中的蛋白质及活性肽

基于鸟枪法蛋白质组学分析方法,使用反相液相色谱-串联质谱（RPLC-MS/MS）系统分析油菜蜂花粉蛋白质的胰蛋白酶酶解产物,结合数据库检索,共鉴定到353条肽段。鉴定到的肽段所归属的蛋白质中有239个蛋白质可检索到其分子生物学功能,主要功能为结合活性、酶活性、运输活性、抑制活性等。根据血管紧张素转化酶（ACE）抑制肽活性与多肽构效之间的关系,从鉴定到的肽段中筛选并适当修饰后得到5条可能具有ACE抑制活性的肽段,化学合成肽段后进行了活性验证。结果表明5条肽段均具有良好的活性,其中肽段AELDIVLALF和LAVNLIPFP表现出较高的ACE抑制活性,半数抑制浓度（IC₅₀）分别为（10.65±0.50）μmol/L和（23.66±1.08）μmol/L。该方法速度快,成本低,大大缩短了鉴定周期,达到了高通量筛选生物活性肽的目的。     

Edman降解中异硫氰酸苯酯新修饰位点的发现和验证

Edman降解是最早建立的一种用于多肽和蛋白质氨基端测序的方法,该方法现在仍被广泛用于生物化学领域。随着高通量蛋白质组学技术的发展和应用,该方法中的异硫氰酸苯酯反应被用于修饰蛋白质氨基端,并用于检测蛋白质水解位点。但还没有异硫氰酸苯酯是否可以修饰其他氨基酸侧链并影响多肽序列分析的研究。为了探究其修饰其他氨基酸的可能性,本文利用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）和液相色谱-串联质谱（LC-MS/MS）研究了异硫氰酸苯酯对一个模型肽的化学修饰。质谱数据解析后发现在高浓度异硫氰酸苯酯的反应条件下,组氨酸上可以引入一个新的异硫氰酸苯酯修饰位点。这一修饰位点的发现预示着通过改变实验条件或分析方法,可以更准确地利用Edman降解和蛋白质组学技术分析多肽和蛋白质。