"De novo" peptide sequencing by MALDI-quadrupole-ion trap mass spectrometry: a preliminary study

Collision-induced dissociation of singly charged peptide ions produced by resonant excitation in a matrix-assisted laser desorption/ionization (MALDI) ion trap mass spectrometer yields relatively low complexity MS/MS spectra that exhibit highly preferential fragmentation, typically occurring adjacent to aspartyl, glutamyl, and prolyl residues. Although these spectra have proven to be of considerable utility for database-driven protein identification, they have generally been considered to contain insufficient information to be useful for extensive de novo sequencing. Here, we report a procedure for de novo sequencing of peptides that uses MS/MS data generated by an in-house assembled MALDI-quadrupole-ion trap mass spectrometer (Krutchinsky, Kalkum, and Chait Anal. Chem. 2001, 73, 5066-5077). Peptide sequences of up 14 amino acid residues in length have been deduced from digests of proteins separated by SDS-PAGE. Key to the success of the current procedure is an ability to obtain MS/MS spectra with high signal-to-noise ratios and to efficiently detect relatively low abundance fragment ions that result from the less favorable fragmentation pathways. The high signal-to-noise ratio yields sufficiently accurate mass differences to allow unambiguous amino acid sequence assignments (with a few exceptions), and the efficient detection of low abundance fragment ions allows continuous reads through moderately long stretches of sequence. Finally, we show how the aforementioned preferential cleavage property of singly charged ions can be used to facilitate the de novo sequencing process.     

Improved protein identification efficiency by mass spectrometry using N-terminal chemical derivatization of peptides from Angiostrongylus costaricensis, a nematode with unknown genome

Matrix-assisted laser desorption ionization (MALDI), Peptide Mass Fingerprinting (PMF) and MALDI-MS/MS ion search (using MASCOT) have become the preferred methods for high-throughput identification of proteins. Unfortunately, PMF can be ambiguous, mainly when the genome of the organism under investigation is unknown and the quality of spectra generated is poor and does not allow confident identification. The post-source decay (PSD) fragmentation of singly charged tryptic peptide ions generated by MALDI-TOF/TOF typically results in low fragmentation efficiency and/or complex spectra, including backbone fragmentation ions (series b and y), internal fragmentation etc. Interpreting these data either manually and/or using de novo sequencing software can frequently be a challenge. To overcome this limitation when studying the proteome of adult Angiostrongylus costaricensis, a nematode with unknown genome, we have used chemical N-terminal derivatization of the tryptic peptides with 4-sulfophenyl isothiocyanate (SPITC) prior to MALDI-TOF/TOF MS. This methodology has recently been reported to enhance the quality of MALDI-TOF/TOF-PSD data, allowing the obtainment of complete sequence of most of the peptides and thus facilitating de novo peptide sequencing. Our approach, consisting of SPITC derivatization along with manual spectra interpretation and Blast analysis, was able to positively identify 76% of analyzed samples, whereas MASCOT analysis of derivatized samples, MASCOT analysis of nonderivatized samples and PMF of nonderivatized samples yielded only 35, 41 and 12% positive identifications, respectively. Moreover, de novo sequencing of SPITC modified peptides resulted in protein sequences not available in NCBInr database paving the way to the discovery of new protein molecules.     

A case study of de novo sequence analysis of N-sulfonated peptides by MALDI TOF/TOF mass spectrometry

The simplicity and sensitivity of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry have increased its application in recent years. The most common method of "peptide mass fingerprint" analysis often does not provide robust identification. Additional sequence information, obtained by post-source decay or collision induced dissociation, provides additional constraints for database searches. However, de novo sequencing by mass spectrometry is not yet common practice, most likely because of the difficulties associated with the interpretation of high and low energy CID spectra. Success with this type of sequencing requires full sequence coverage and demands better quality spectra than those typically used for data base searching. In this report we show that full-length de novo sequencing is possible using MALDI TOF/TOF analysis. The interpretation of MS/MS data is facilitated by N-terminal sulfonation after protection of lysine side chains (Keough et al., Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 7131-7136). Reliable de novo sequence analysis has been obtained using sub-picomol quantities of peptides and peptide sequences of up to 16 amino acid residues in length have been determined. The simple, predictable fragmentation pattern allows routine de novo interpretation, either manually or using software. Characterization of the complete primary structure of a peptide is often hindered because of differences in fragmentation efficiencies and in specific fragmentation patterns for different peptides. These differences are controlled by various structural parameters including the nature of the residues present. The influence of the presence of internal Pro, acidic and basic residues on the TOF/TOF fragmentation pattern will be discussed, both for underivatized and guanidinated/sulfonated peptides.     

A novel MALDI LIFT-TOF/TOF mass spectrometer for proteomics

A new matrix-assisted laser-desorption/ionization time-of-flight/time-of-flight mass spectrometer with the novel "LIFT" technique (MALDI LIFT-TOF/TOF MS) is described. This instrument provides high sensitivity (attomole range) for peptide mass fingerprints (PMF). It is also possible to analyze fragment ions generated by any one of three different modes of dissociation: laser-induced dissociation (LID) and high-energy collision-induced dissociation (CID) as real MS/MS techniques and in-source decay in the reflector mode of the mass analyzer (reISD) as a pseudo-MS/MS technique. Fully automated operation including spot picking from 2D gels, in-gel digestion, sample preparation on MALDI plates with hydrophilic/hydrophobic spot profiles and spectrum acquisition/processing lead to an identification rate of 66% after the PMF was obtained. The workflow control software subsequently triggered automated acquisition of multiple MS/MS spectra. This information, combined with the PMF increased the identification rate to 77%, thus providing data that allowed protein modifications and sequence errors in the protein sequence database to be detected. The quality of the MS/MS data allowed for automated de novo sequencing and protein identification based on homology searching.     

Improved protein identification efficiency by mass spectrometry using N-terminal chemical derivatization of peptides from Angiostrongylus costaricensis, a nematode with unknown genome

Matrix-assisted laser desorption ionization (MALDI), Peptide Mass Fingerprinting (PMF) and MALDI-MS/MS ion search (using MASCOT) have become the preferred methods for high-throughput identification of proteins. Unfortunately, PMF can be ambiguous, mainly when the genome of the organism under investigation is unknown and the quality of spectra generated is poor and does not allow confident identification. The post-source decay (PSD) fragmentation of singly charged tryptic peptide ions generated by MALDI-TOF/TOF typically results in low fragmentation efficiency and/or complex spectra, including backbone fragmentation ions (series b and y), internal fragmentation etc. Interpreting these data either manually and/or using de novo sequencing software can frequently be a challenge. To overcome this limitation when studying the proteome of adult Angiostrongylus costaricensis, a nematode with unknown genome, we have used chemical N-terminal derivatization of the tryptic peptides with 4-sulfophenyl isothiocyanate (SPITC) prior to MALDI-TOF/TOF MS. This methodology has recently been reported to enhance the quality of MALDI-TOF/TOF-PSD data, allowing the obtainment of complete sequence of most of the peptides and thus facilitating de novo peptide sequencing. Our approach, consisting of SPITC derivatization along with manual spectra interpretation and Blast analysis, was able to positively identify 76% of analyzed samples, whereas MASCOT analysis of derivatized samples, MASCOT analysis of nonderivatized samples and PMF of nonderivatized samples yielded only 35, 41 and 12% positive identifications, respectively. Moreover, de novo sequencing of SPITC modified peptides resulted in protein sequences not available in NCBInr database paving the way to the discovery of new protein molecules.     

CHASE, a charge-assisted sequencing algorithm for automated homology-based protein identifications with matrix-assisted laser desorption/ionization time-of-flight post-source decay fragmentation data

We describe CHASE, a novel algorithm for automated de novo sequencing based on the mass spectrometric (MS) fragmentation analysis of tryptic peptides. This algorithm is used for protein identification from sequence similarity criteria and consists of four steps: (1) derivatization of tryptic peptides at the N-terminus with a negatively charged reagent; (2) post-source decay (PSD) fragmentation analysis of peptides; (3) interpretation of the mass peaks with the CHASE algorithm and reconstruction of the amino acid sequence; (4) transfer of these data to software for protein identifications based on sequence homology (Basic Local Alignment Search Tool, BLAST). This procedure deduced the correct amino acid sequence of tryptic peptide samples and also was able to deduce the correct sequence from difficult mass patterns and identify the amino acid sequence. This allows complete automation of the process starting from MS fragmentation of complex peptide mixtures at low concentration (e.g. from silver-stained gel bands) to identification of the protein. We also show that if PSD data are collected in a single spectrum (instead of the segmented mode offered by conventional matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) instrumentation), the complete workflow from MS-PSD data acquisition to similarity-based identification can be completely automated. This strategy may be applied to proteomic studies for protein identification based on automated de novo sequencing instead of MS or tandem MS patterns. We describe the Charge Assisted Sequencing Engine (CHASE) algorithm, the working protocol, the performance of the algorithm on spectra from MALDI-TOFMS and the data comparison between a TOF and a TOF-TOF instrument.     

Isolation and sequence analysis of peptides from the skin secretion of the Middle East tree frog Hyla savignyi

Novel peptides were identified in the skin secretion of the tree frog Hyla savignyi. Skin secretions were collected by mild electrical stimulation. Peptides were separated by reversed-phase high-performance liquid chromatography. Mass spectra were acquired by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), and fragment ion spectra were obtained after collision-induced dissociation and electron capture dissociation. Peptides were analyzed by manual de novo sequencing and composition-based sequencing (CBS). Sequence analyses of three so far undescribed, structurally unrelated peptides are presented in this paper, having the sequences DDSEEEEVE-OH, P*EEVEEERJK-OH, and GJJDPJTGJVGGJJ-NH₂. The glutamate-rich sequences are assumed to be acidic spacer peptides of the prepropeptide. One of these peptides contains the modified amino acid hydroxyproline, as identified and localized by high-accuracy FTICR-MS. Combination of CBS and of experience-based manual sequence analysis as complementary and database-independent sequencing strategies resulted in peptide identification with high reliability.

白眉蝮蛇蛇毒及蛇毒酶的激光解吸飞行时间质谱研究

Perkins等[1]用MALDI/TOF/MS对不同种类蛇毒中神经毒素进行了分析,彭嘉柔等[2,3]对江浙蝮蛇和蛇毒粗组份进行了初步质谱表征.但蛇毒蛋白纯化困难,因此对单一组份的研究较少且不系统.本文以白眉蝮蛇蛇毒(AgkistrodonblomhoffiiUssurensis,ABUV)为原料,纯化得到了精氨酸酯酶(Arginineesterase,AEase)、磷脂酶A2(PhospholipaseA2,PLA2)、纤溶酶(fibrinolyticenzyme)和L-氨基酸氧化酶(L-aminoacidoxidase),并且用MALDI/TOF/MS法对它们和蛇毒粗毒进行了系统研究.1　实验部分1.1　仪器和药品　激光解吸质谱仪为美国Molecular公司L…     

Separation and analysis of peptides and proteins from Vipera lebetina snake venom

Our studies of Levantine viper venom have demonstrated that the venom is a rich source of biomedically important proteins and peptides. The venom contains metalloproteases: thrombolytic, fibrin-degrading lebetase, an endothelial cell apoptosis inducing metalloprotease (VLAIP), factor X activator (VLFXA); serine proteases: factor V activator, bradykinin-releasing serine proteases, β-fibrinogenase, α-fibrinogenase and chymotrypsin-like protease and different other enzymes such as phosphodiesterase, 5`-nucleotidase, ribonuclease, phospholipase A2s and L-amino acid oxidase. Among nonenzymatic components venom contains: nerve growth factor, vascular endothelial growth factor, disintegrins, C-type lectins.Here we report the isolation and characterization of proteins and peptides from Vipera lebetina venom using size exclusion, ion exchange, hydrophobic interaction and affinity chromatography, HPLC, UPLC and MALDI-TOF MS methods. N-terminal sequences and internal sequences of tryptic peptides of different proteins were determined using Edman sequencing and LC-ESI-MS/MS techniques. On the basis of fragmental sequences of proteins the oligonucleotides were designed and used as primers for cDNA cloning. Using cDNA library of the venom gland of a single snake the cDNAs coding proteins were cloned and sequenced. Protein sequences were deduced from cDNA sequences.The substrate specificity of venom proteases against insulin B-chain, bradykinin, substance P, and 6-10 amino acid residues containing peptides synthesized according to potential cleavage regions of fibrinogen, factor X, factor IX, factor V, α₂-macroglobulin bait region and pregnancy zone protein were studied using MALDI-TOF mass spectrometry technique.     

The nature of collision-induced dissociation processes of doubly protonated peptides: comparative study for the future use of matrix-assisted laser desorption/ionization on a hybrid quadrupole time-of-flight mass spectrometer in proteomics.

Comparative MS/MS studies of singly and doubly charged electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) precursor peptide ions are described. The spectra from these experiments have been evaluated with particular emphasis on the data quality for subsequent data processing and protein/amino acid sequence identification. It is shown that, once peptide ions are formed by ESI or MALDI, their charge state, as well as the collision energy, is the main parameter determining the quality of collision-induced dissociation (CID) MS/MS fragmentation spectra of a given peptide. CID-MS/MS spectra of singly charged peptides obtained on a hybrid quadrupole orthogonal time-of-flight mass spectrometer resemble very closely spectra obtained by matrix-assisted laser desorption/ionization post-source decay time-of-flight mass spectrometry (MALDI-PSD-TOFMS). On the other hand, comparison of CID-MS/MS spectra of either singly or doubly charged ion species shows no dependence on whether ions have been formed by ESI or MALDI. This observation confirms that, at the time of precursor ion selection, further mass analysis is effectively decoupled from the desorption/ionization event. Since MALDI ions are predominantly formed as singly charged species and ESI ions as doubly charged, the associated difference in the spectral quality of MS/MS spectra as described here imposes direct consequences on data processing, database searching using ion fragmentation data, and de novo sequencing when ionization techniques are changed.     

PEAKS: powerful software for peptide de novo sequencing by tandem mass spectrometry

A number of different approaches have been described to identify proteins from tandem mass spectrometry (MS/MS) data. The most common approaches rely on the available databases to match experimental MS/MS data. These methods suffer from several drawbacks and cannot be used for the identification of proteins from unknown genomes. In this communication, we describe a new de novo sequencing software package, PEAKS, to extract amino acid sequence information without the use of databases. PEAKS uses a new model and a new algorithm to efficiently compute the best peptide sequences whose fragment ions can best interpret the peaks in the MS/MS spectrum. The output of the software gives amino acid sequences with confidence scores for the entire sequences, as well as an additional novel positional scoring scheme for portions of the sequences. The performance of PEAKS is compared with Lutefisk, a well-known de novo sequencing software, using quadrupole-time-of-flight (Q-TOF) data obtained for several tryptic peptides from standard proteins.     

金属离子对蛇毒蛋白生物活性及结构效应的影响

从旅顺产白眉蝮蛇(Gloydius blomhoffii brevicaudus, GBB) 蛇毒中纯化得到了3种电泳和质谱纯蛋白活性组分, 其酶解肽段采用高效液相色谱-电喷雾串联质谱(HPLC-nESI-MS/MS)进行序列测定, 与其它同源性蛇毒蛋白氨基酸序列比对发现, 3种蛋白为新的蛇毒磷脂酶A₂、类凝血酶和金属蛋白酶, 分别将其命名为GBB-bPLA₂, GBB-TLE和GBB-MP. 电感耦合等离子体发射光谱法(ICP-AES) 测得每个GBB-bPLA₂和GBB-MP中含有一个Ca²⁺; 每个GBB-TLE中含有2个Zn²⁺. Ca²⁺可分别使GBB-bPLA₂和GBB-MP的荧光发射波长向短波方向移动2.0和1.6 nm, 使二者的荧光发射强度提高14.0%和11.0%; Ca²⁺的存在可显著提高二者的热稳定性, 使GBB-bPLA₂和GBB-MP的热变性温度分别提高1.5和2.0 ℃. Zn²⁺使GBB-TLE的荧光发射强度增高4.3%, 但对GBB-TLE的酯酶水解活性、荧光发射波长和热变性温度无显著影响. 金属离子的存在能够不同程度地影响蛇毒蛋白结构热稳定性, 但对蛇毒蛋白生物活性的作用则不同.     

A Quantitative Tool to Distinguish Isobaric Leucine and Isoleucine Residues for Mass Spectrometry-Based De Novo Monoclonal Antibody Sequencing

De novo sequencing by mass spectrometry (MS) allows for the determination of the complete amino acid (AA) sequence of a given protein based on the mass difference of detected ions from MS/MS fragmentation spectra. The technique relies on obtaining specific masses that can be attributed to characteristic theoretical masses of AAs. A major limitation of de novo sequencing by MS is the inability to distinguish between the isobaric residues leucine (Leu) and isoleucine (Ile). Incorrect identification of Ile as Leu or vice versa often results in loss of activity in recombinant antibodies. This functional ambiguity is commonly resolved with costly and time-consuming AA mutation and peptide sequencing experiments. Here, we describe a set of orthogonal biochemical protocols, which experimentally determine the identity of Ile or Leu residues in monoclonal antibodies (mAb) based on the selectivity that leucine aminopeptidase shows for n-terminal Leu residues and the cleavage preference for Leu by chymotrypsin. The resulting observations are combined with germline frequencies and incorporated into a logistic regression model, called Predictor for Xle Sites (PXleS) to provide a statistical likelihood for the identity of Leu at an ambiguous site. We demonstrate that PXleS can generate a probability for an Xle site in mAbs with 96% accuracy. The implementation of PXleS precludes the expression of several possible sequences and, therefore, reduces the overall time and resources required to go from spectra generation to a biologically active sequence for a mAb when an Ile or Leu residue is in question.

Sequencing of Oligourea Foldamers by Tandem Mass Spectrometry

This study is focused on sequence analysis of peptidomimetic helical oligoureas by means of tandem mass spectrometry, to build a basis for de novo sequencing for future high-throughput combinatorial library screening of oligourea foldamers. After the evaluation of MS/MS spectra obtained for model compounds with either MALDI or ESI sources, we found that the MALDI-TOF-TOF instrument gave more satisfactory results. MS/MS spectra of oligoureas generated by decay of singly charged precursor ions show major ion series corresponding to fragmentation across both CO-NH and N′H-CO urea bonds. Oligourea backbones fragment to produce a pattern of a, x, b, and y type fragment ions. De novo decoding of spectral information is facilitated by the occurrence of low mass reporter ions, representative of constitutive monomers, in an analogous manner to the use of immonium ions for peptide sequencing.      

A new strategy utilizing electrospray ionization-quadrupole ion trap mass spectrometry for the qualitative determination of GnRH peptides

Numerous forms of the neurotransmitter GnRH have been discovered in vertebrates and invertebrates. Methods used for identification of these peptides are laborious and often require the application of multiple, confirmatory techniques. In this study, we investigate whether HPLC-MS/MS and de novo sequencing techniques applied to whole peptide analysis can provide a simpler approach to GnRH characterization. Experiments were performed with six GnRH forms (chicken I, chicken II, lamprey III, mammalian, salmon and seabream) to determine whether MS/MS spectra would be dominated by proline-directed fragmentation to the detriment of obtaining sufficient fragmentation for sequencing. While the expected b8 fragment was prominent, sufficient ion series were obtained for the six GnRH peptides to provide sequence identification. On the basis of the patterns observed for six model peptides, similar fragmentation patterns are expected for other GnRH forms. To confirm the applicability of the method, extracts from Sprague-Dawley rat brains were examined. These experiments confirm the presence of mammalian GnRH and a posttranslationally modified form of mammalian GnRH, hydroxyproline9 GnRH, in Sprague-Dawley rat brains and demonstrate that ESI-MS/MS techniques provide a valuable addition to existing qualitative methods.     

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。     

Composite sequence proteomic analysis of protein biomarkers of Campylobacter coli, C. lari and C. concisus for bacterial identification

Protein biomarkers observed in the matrix-assisted laser desorption/ionization time-of-flight mass spectra (MALDI-TOF-MS) of cell lysates of three strains of Campylobacter coli, two strains of C. lari and one strain of C. concisus have been identified by 'bottom-up' proteomic techniques. The significant findings are as follows. First, the protein biomarkers identified were: PhnA-related protein, 4-oxalocrotonate tautomerase (DmpI)-related protein, NifU-like protein, cytochrome c, DNA-binding protein HU, 10 kDa chaperonin, thioredoxin, as well as several conserved hypothetical and ribosomal proteins. Second, variations in the biomarker ion m/z in MALDI-TOF-MS spectra across species and strains are the result of variations in the amino acid sequence of the protein due to non-synonymous mutations of the biomarker gene. Third, the most common post-translational modifications (PTMs) were the removal of the N-terminal methionine and N-terminal signal peptides. However, in the case of the NifU protein (an iron-sulfur cluster transport protein), post-translational cleavage occurred from the C-terminus. Fourth, only the genomes of the C. coli strain RM2228 and C. lari strain RM2100 have been sequenced; thus, proteomic identification of the proteins of the other strains in this study relied upon sequence homology to the genomic sequence of these strains as well as the genomes of sequences of other Campylobacter strains. In some cases, the determination of the full amino acid sequence of a protein biomarker from a genomically non-sequenced strain was accomplished by combining non-overlapping partial sequences from proteomic identifications of genomically-sequenced strains that were of the same species (or of a different species) to that of the non-sequenced strain. The accuracy of this composite sequence was confirmed by both MS and MS/MS. It was necessary, in some cases, to perform de novo sequencing on 'gaps' in the composite sequence that were not homologous to any genomically-sequenced strain. In order to validate the composite sequence approach, composite sequences were further confirmed by subsequent DNA sequencing of the biomarker gene. Thus, using the composite sequence approach, it was possible to determine the full amino acid sequence of an unknown protein from a genomically non-sequenced bacterial strain without the necessity of either sequencing the biomarker gene or performing full de novo MS/MS sequencing. The sequence obtained could then be used as a strain-specific biomarker for analysis by 'top-down' proteomics techniques.     

Application of the ETD/PTR reactions in top‐down proteomics as a faster alternative to bottom‐up nanoLC‐MS/MS protein identification

Our goal was to compare two popular analytical techniques used nowadays in proteomic investigations for proteins/peptides sequencing and identification, a widely used nanoLC‐MS/MS approach applied in the bottom‐up proteomics and electron transfer dissociation/proton transfer reaction fragmentation preferably used when top‐down strategy is applied. Comparison was carried out with the aid of the ESI‐quadrupole ion‐trap instrument using the following criteria: total time of analysis including sample preparation, sequence coverage, Mascot scoring, capability to detect modifications, quality of the results as a function of protein molecular weight and sample consumption. Copyright © 2012 John Wiley & Sons, Ltd.     

Formation of c1 fragment ions in collision-induced dissociation of glutamine-containing peptide ions: a tip for de novo sequencing

A c1 ion was observed with significant yield in the tandem mass (MS/MS) spectra of peptide ions containing glutamine as the second amino acid residue from the N-terminus. The c1 fragment was generated independently of the N-terminal residue of the peptide, but its abundance was strongly dependent on the side-chain identity. This ion is not a common fragmentation product in low-energy collision-induced dissociation of peptide ions, but it assists in identification of the first two amino acid residues, often difficult due to a low or absent signal from the heaviest y ion. A consecutive fragmentation mechanism is proposed, involving a b2 ion with a six-membered ring as an intermediate, to explain the exceptional stability of the c1 fragment ion. The utility of this information is discussed, especially in de novo sequencing of peptide ions.     

马氏钳蝎蝎毒短肽BmK622的分离纯化和一级结构测定

应用凝胶过滤、离子交换和HPLC反相色谱法从马氏蝎粗毒中分离纯化得到蝎毒 多肽BmK622．联合运用串联质谱法和Edman降解法，鉴定了KmK622N端19个残基的序 列，经过数据库检索，发现数据库中用cDNA克隆方法鉴定了序列的马氏钳蝎蝎毒短 肽BmTX3一级序列N端19个残基与BmK622已测定的N端19个残基序列完全相同， BmK622的分子量测定和氨基酸组成分折的结果表明，BmK22与BmTX3分子量相同、氨 基酸组成一致，从而BmK22的一级结构为：GFLID VKCFA SSECW TACKK VTGSG QGKCQ NNQCR CY．