C-terminal sequence determination of peptides degraded with carboxypeptidases of different specificities and analyzed by 252-Cf plasma desorption mass spectrometry

Identification of the truncated peptides by plasma desorption mass spectrometry in C-terminal sequence determination with carboxypeptidases offers several advantages over analysis of the liberated amino acids. It is possible to perform in situ digestion of a nitrocellulose-bound sample already used for molecular weight determination and thus obtain sequence information without further sample consumption. In time-course analysis the analytical information, although not obtained in real time, is sufficiently rapid to adjust the digestion conditions. There is no need for quantitation because the identification is based on molecular weight differences. Sensitivity in the low picomole range is obtainable. The digestion of a number of peptides (900-3500 Da) with carboxypeptidase Y and MII has been monitored. It was found that successive use of the enzymes or use of a mixture of the enzymes was often advantageous. The sequence of up to 10 residues from the C-terminus has been determined for the peptides studied.     

Nanoporous surfaces as harvesting agents for mass spectrometric analysis of peptides in human plasma

Silica-based nanoporous surfaces have been developed in order to capture low molecular weight peptides from human plasma. Harvested peptides were subjected to mass spectrometric analysis by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a means of detecting and assessing the bound molecules. Peptide profiles consisting of about 70 peaks in the range 800-10,000 m/z were generated. The method could allow detection of small peptides at ng/mL concentration levels, either in standard solutions or in plasma. The same molecular cutoff effect was observed for mixtures of standard proteins and peptides incubated with silicon-based nanoporous surfaces.     

Mass spectrometric identification of serum peptides employing derivatized poly(glycidyl methacrylate/divinyl benzene) particles and mu-HPLC

Biomarkers play a key role in preclinical screening and diagnosis of a disease. Various support materials are utilized for this task, in combination with MALDI-TOF-MS. The way to effectively bind serum contents and their profiling is well-elaborated by the material-enhanced laser desorption ionization (MELDI) approach. In this particular work, focus is placed on the development of a strategy to identify low molecular weight serum peptides. Poly(GMA/DVB) is derivatized in a way to achieve an affinity termed as immobilized metal ion affinity chromatography (IMAC). Iminodiacetic acid (IDA) is used as a chelating ligand, whereas copper (Cu2+) acts as a metal ion for complexing peptides and proteins out of blood serum. Polymer binds the serum compounds over a broad mass range, which includes low mass peptides and high mass albumin (66 kDa). Bound contents are eluted from material by an acetonitrile/trifluoroacetic acid mixture, which proves the reversible nature of metal and amino acid linkage. Polystyrene/divinyl benzene (PS/DVB) monolithic capillary column is used for fractionation through RP-HPLC, prior to the target spotting. The tandem TOF fragment ion mass spectra of each fraction is acquired and used to search against the Swiss-Prot database, using the Mascot search engine for the identification of peptides.     

Comprehensive profiling of the low molecular weight proteins and peptides in weak cation exchange beads human serum retentate

Mass spectrometric profiling using ProteinChip and magnetic beads has rapidly grown over the past years, particularly to generate serum profiles for cancer diagnosis. The molecular weights of these distinguishing peaks are usually under 30 kDa. To identify those low molecular weight proteins and peptides is important for specific assays to be developed and increases biological insight. In this study, low molecular weight proteins and peptides from serum were purified by a combination of weak cation exchange magnetic beads and high performance liquid chromatography. The purified proteins and peptides were analyzed by 1D SDS PAGE, SELDI and LC-MS/MS. 246 proteins were identified from the HPLC fractions by LC-MS/MS. 95(38.62%) proteins were first identified in serum compare with Sys-BodyFluid database. 11(11/96) proteins were documented cancer associated proteins. We also observed about 109 proteins/peptides in SELDI mass spectrum, and 13 of the SELDI features were identified.     

Identification of disulfide-containing peptides by performic acid oxidation and mass spectrometry

In addition to reducing the analysis time, the direct examination of proteolytic digests by fast atom bombardment mass spectrometry (FABMS) greatly extends the information that is available from peptide mapping experiments. Mass spectral data are particularly useful for identifying post-translationally modified peptides. For example, the molecular weight of a disulfide-containing peptide may be used to locate the disulfide bond in the protein from which the peptide was derived. This paper describes a new procedure, which is useful for identifying disulfide-bonded peptides. Peptides are treated with performic acid to modify certain residues and thereby cause a characteristic change in the peptide molecular weight. This change in molecular weight is determined by FABMS and used to help identify peptides. Results for a series of small peptides demonstrate that Cys, Met, and Trp are the only residues that undergo a change in molecular weight under the conditions used here. Furthermore, these changes in molecular weight are diagnostic for each of the residues. Cysteinyl-containing peptides are of particular interest, because their identification is essential for locating disulfide bonds. The molecular weight of a peptide increases by 48 mu for each cysteinyl residue present. This approach is used to identify peptides that contain both cysteinyl and cystinyl residues in the peptic digest of bovine insulin. The method is extended to the analysis of a tryptic digest of cyanogen bromide-treated ribonuclease A. A computer-assisted analysis procedure is used to demonstrate the specificity with which peptide molecular weight is related to specific segments of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct tandem mass spectrometry reveals limitations in protein profiling experiments for plasma biomarker discovery

The low molecular weight plasma proteome and its biological relevance are not well defined; therefore, experiments were conducted to directly sequence and identify peptides observed in plasma and serum protein profiles. Protein fractionation, matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) profiling, and liquid-chromatography coupled to MALDI tandem mass spectrometry (MS/MS) sequencing were used to analyze the low molecular weight proteome of heparinized plasma. Four fractionation techniques using functionally derivatized 96-well plates were used to extract peptides from plasma. Tandem TOF was successful for identifying peptides up to m/z 5500 with no prior knowledge of the sequence and was also used to verify the sequence assignments for larger ion signals. The peptides (n>250) sequenced in these profiles came from a surprisingly small number of proteins (n approximately 20), which were all common to plasma, including fibrinogen, complement components, antiproteases, and carrier proteins. The cleavage patterns were consistent with those of known plasma proteases, including initial cleavages by thrombin, plasmin and complement proteins, followed by aminopeptidase and carboxypeptidase activity. On the basis of these data, we discuss limitations in biomarker discovery in the low molecular weight plasma or serum proteome using crude fractionation coupled to MALDI-MS profiling.     

颜氏大疣蛛蛛毒中多肽和蛋白质的多样性分析

对颜氏大疣蛛Macrothele yani蛛毒所富含的多肽与蛋白质多样性进行探索,采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和超高效液相色谱-电喷雾-四极杆-飞行时间质谱技术分离和鉴定颜氏大疣蛛蛛毒中的蛋白质和多肽,并对其相对分子质量分布多样性进行分析。结果显示:粗毒中所含蛋白质的相对分子质量主要分布在35kDa以上。在17~135kDa分离度较佳的共有11条电泳条带,主要集中于40~120kDa附近;在75kDa附近弥散着高丰度的蛋白条带,75kDa以上的蛋白质最丰富。粗毒经色谱分离后得到超过50个色谱峰,经质谱鉴定得到121个物质成分,其中,多肽类物质的相对分子质量呈双峰式分布,21%分布在500~2000 Da,76%分布在3000~5000Da,为粗毒中多肽含量最丰富的部分,且集中于35~60min的保留时间内被洗脱。研究结果表明,颜氏大疣蛛蛛毒中含有较为丰富的多肽和蛋白类物质,这些物质的相对分子质量分布特征与已报道的其他蜘蛛既有相似性又存在具体差异。本文展示了颜氏大疣蛛蛛毒的分子多样性,为后续该毒素的物质基础研究及药用价值开发提供参考。     

Peptide studies using a fast atom bombardment high field mass spectrometer and data system. 4. Disulfide-containing peptides

Ten peptides containing one, two or three disulfides were examined to determine their behavior under fast atom bombardment (FAB) mass spectrometric conditions. The mass spectra for the disulfide and the reduced disulfide forms of each peptide were compared. Several factors were examined that contribute to the fast atom bombardment mass spectra of these peptides: components of the FAB matrix such as alkali cations, acids, bases and reducing agents, the intrinsic molecular properties of the intact peptide, and the effect of reducing conditions on sensitivity. The FAB mass spectra of the disulfide-containing peptides examined in this study displayed accurate molecular weight information and fragmentation which indicated the position of the disulfide in the amino acid sequence.     

The primary structure of thioredoxin from Chromatium vinosum determined by high-performance tandem mass spectrometry

The primary structure of thioredoxin, a redox protein isolated from Chromatium vinosum, was determined by high-performance tandem mass spectrometry, which permitted sequencing of the 14 peptides (ranging in length from 2 to 18 amino acids) generated by digestion with trypsin and of several peptides produced by Staphylococcus aureus protease. The mass spectrometrically determined molecular weights of the peptides from the latter digest were used to properly align the tryptic peptides, which could also be accomplished on the basis of the considerable homology with Escherichia coli thioredoxin. Finally, the molecular weight of the Chromatium thioredoxin was determined by mass spectrometry and found to be 11,748.0, in good agreement with 11,750.2 calculated for the proposed sequence. Although it was difficult to establish by mass spectrometry, five leucines and three isoleucines could be identified, leaving only eight undifferentiated.     

Application of the dansylation reaction to the characterization of low molecular weight peptides by dodecyl sulfate-polyacrylamide-gel electrophoresis.

Polyacrylamide-gel electrophoresis of low molecular weight DNS-peptides was performed in the presence of 0.1% sodium dodecyl sulfate and 8 m urea. This procedure enabled an estimation of the molecular weight of peptides at the nanomole level without staining. A linear relationship between molecular weight and mobility was obtained over a molecular weight range of 1,000–12,000, although a few anomalous peptides (e.g., glucagon and insulin) and small peptides of molecular weight less than 1,000 deviated from linearity. The N-terminal amino acid of a peptide can be determined in combination with thin layer chromatography on polyamide sheets. The usefulness of this procedure for checking small amounts of contaminant in an oligopeptide sample was also noted.     

Comprehensive peptidome analysis of mouse livers by size exclusion chromatography prefractionation and nanoLC-MS/MS identification

Peptidome analysis has received increasing attention in recent years. Cancer diagnosis by serum peptidome has also been reported by peptides' profiling for discovery of peptide biomarkers. Tissue, which may have a higher biomarker concentration than blood, has not been investigated extensively by means of peptidome analysis. Here, a method for the peptidome analysis of mouse liver was developed by the combination of size exclusion chromatography (SEC) prefractionation with nano-liquid chromatography-tamdem mass spectrometry (nanoLC-MS/MS) analysis. The extracted peptides from mouse liver were separated according to their molecular weight using a size exclusion column. MALDI-TOF MS was used to characterize the molecular weight distribution of the peptides in fractions eluted from the SEC column. The low molecular weight (LMW) (MW < 3000 Da) peptides in the collected fractions were directly analyzed by LC-MS/MS which resulted in the identification of 1181 unique peptides (from 371 proteins). The high molecular weight (HMW) (MW > 3000 Da) peptides in the early two fractions from the SEC column were first digested with trypsin, and the resulted digests were then analyzed by LC-MS/MS, which led to the identification of 123 and 127 progenitor proteins of the HMW peptides in fractions 1 and 2, respectively. Analysis of the peptides' cleavage sites showed that the peptides are cleaved in regulation, which may reflect the protease activity and distribution in body, and also represent the biological state of the tissue and provide a fresh source for biomarker discovery.     

基于串联质谱的鱼皮明胶鉴别研究

在胶原蛋白序列比对基础上,以虹鳟鱼明胶、猪明胶和牛明胶为模型,利用高效液相色谱－串联质谱技术（HPLC-MS/MS）研究了3种明胶降解多肽组成的差异。使用胰蛋白酶将鱼皮明胶进行了酶解处理,使用HPLC-MS/MS对酶解产物中的多肽组成进行了分析,并与猪和牛明胶酶解产物中的多肽进行了比较。结果表明鱼明胶酶解产物中存在特征多肽,通过特征多肽的种类可区别鱼明胶与猪和牛明胶,研究了明胶多肽中脯氨酸羟基化修饰、明胶分子量范围和脱酰胺化对特征多肽识别的影响。研究表明利用HPLC-MS/MS技术通过识别明胶酶解产物中的特征多肽进行鱼皮明胶鉴别具有可行性。     

"Top Down" characterization is a complementary technique to peptide sequencing for identifying protein species in complex mixtures

At present, mass spectrometry provides a rapid and sensitive means for making conclusive protein identifications from complex mixtures. Sequencing tryptic peptides derived from proteolyzed protein samples, also known as the "Bottom Up" approach, is the mass spectrometric gold standard for identifying unknowns. An alternative technology, "Top Down" characterization, is emerging as a viable option for protein identifications, which involves analyzing the intact unknowns for accurate mass and amino acid sequence tags. In this paper, both characterization methods were employed to more comprehensively differentiate two early-eluting peaks in a process-scale size-exclusion chromatography (SEC) step for a recombinant, immunoglobulin gamma-1 (IgG-1) fusion protein. The contents of each SEC peak were enzymatically digested, and the resulting peptides were mapped using reversed-phase (RP) HPLC-ion trap MS. Many low-level UV signals were observed among the fusion protein-related peptide peaks. These unknowns were collected, concentrated, and analyzed using nanoelectrospray (nanoES) collision-induced dissociation (CID) tandem (MS/MS) mass spectrometry for identification. The peptide sequencing experiments resulted in the identification of twenty host cell-related proteins. Following peptide mapping, the contents of the two SEC peaks were protein mass profiled using on-line RP HPLC coupled to a high-resolution, quadrupole time-of-flight (Qq/TOF) MS. Unknown proteins were also collected, concentrated, and dissociated using nanoES CID MS/MS. Intact protein CID experiments and accurate molecular weight information allowed for the identification of three full length host cell-derived proteins and numerous clips from these and additional proteins. The accurate molecular weight values allowed for the assignment of N- and C-terminal processing, which is difficult to conclusively access from peptide mapping data. The peptide-mapping experiments proved to be far more effective for making protein identifications from complex mixtures, whereas the protein mass profiling was useful for assessing modifications and distinguishing protein clips from full length species.     

Characterization of the low molecular weight human serum proteome

Serum potentially carries an archive of important histological information whose determination could serve to improve early disease detection. The analysis of serum, however, is analytically challenging due to the high dynamic concentration range of constituent protein/peptide species, necessitating extensive fractionation prior to mass spectrometric analyses. The low molecular weight (LMW) serum proteome is that protein/peptide fraction from which high molecular weight proteins, such as albumin, immunoglobulins, transferrin, and lipoproteins, have been removed. This LMW fraction is made up of several classes of physiologically important proteins such as cytokines, chemokines, peptide hormones, as well as proteolytic fragments of larger proteins. Centrifugal ultrafiltration of serum was used to remove the large constituent proteins resulting in the enrichment of the LMW proteins/peptides. Because albumin is known to bind and transport small molecules and peptides within the circulatory system, the centrifugal ultrafiltration was conducted under solvent conditions effecting the disruption of protein-protein interactions. The LMW serum proteome sample was digested with trypsin, fractionated by strong cation exchange chromatography, and analyzed by microcapillary reversed-phase liquid chromatography coupled on-line with electrospray ionization tandem mass spectrometry. Analysis of the tandem mass spectra resulted in the identification of over 340 human serum proteins; however, not a single peptide from serum albumin was observed. The large number of proteins identified demonstrates the efficacy of this method for the removal of large abundant proteins and the enrichment of the LMW serum proteome.     

A novel quantitative proteomics workflow by isobaric terminal labeling

Quantification by series of b, y fragment ion pairs generated from isobaric-labeled peptides in MS2 spectra has recently been considered an accurate strategy in quantitative proteomics. Here we developed a novel MS2 quantification approach named quantitation by isobaric terminal labeling (QITL) by coupling (18)O labeling with dimethylation. Trypsin-digested peptides were labeled with two (16)O or (18)O atoms at their C-termini in H(2)(16)O or H(2)(18)O. After blocking all ε-amino groups of lysines through guanidination, the N-termini of the peptides were accordingly labeled with formaldehyde-d(2) or formaldehyde. These indistinguishable, isobaric-labeled peptides in MS1 spectra produce b, y fragment ion pairs in the whole mass range of MS2 spectra that can be used for quantification. In this study, the feasibility of QITL was first demonstrated using standard proteins. An accurate and reproducible quantification over a wide dynamic range was achieved. Then, complex rat liver samples were used to verify the applicability of QITL for large-scale quantitative analysis. Finally, QITL was applied to profile the quantitative proteome of hepatocellular carcinoma (HCC) and adjacent non-tumor liver tissues. Given its simplicity, low-cost, and accuracy, QITL can be widely applied in biological samples (cell lines, tissues, and body fluids, etc.) for quantitative proteomic research.     

High performance liquid chromatography and time-of-flight secondary ion mass spectrometry: a new dimension in structural analysis of apolipoproteins

We report the isolation and characterization of an apolipoprotein A-I mutant using a new technique for structural analysis of apolipoproteins based upon the combined techniques of protein isolation by isoelectric focusing in immobilized pH-gradients, reversed-phase HPLC of tryptic peptides, and subsequent molecular weight analysis of isolated peptides by time-of-flight secondary ion mass spectrometry (TOF-SIMS). The particular advantages of the TOF-SIMS procedure in the characterization of proteolytic peptides are the detection limits in the picomole range, the accuracy of molecular weight determination (up to 3000 +/- 1 D), the speed of analysis, and the wide range of applications for involatile biomolecules. The described procedure for the analysis of apolipoproteins requires only 2 ml of serum as starting material. This method can be used to monitor for genetic polymorphisms and posttranslational modifications on a microscale basis. Applying these techniques, we characterized a new apolipoprotein A-I mutant with an amino acid exchange arginine177 by histidine.     

Primary structure of Paim I, an alpha-amylase inhibitor from Streptomyces corchorushii, determined by the combination of Edman degradation and fast atom bombardment mass spectrometry

Paim I, a protein alpha-amylase inhibitor, inhibits animal alpha-amylases from pig, dog, cow, horse, etc. but has no activity against human salivary and pancreatic amylases. The primary structure of Paim I has been determined by Edman degradation and fast atom bombardment mass spectrometry (FABMS). This protein is a single-chain polypeptide of 73 amino acid residues with a calculated molecular weight from the sequence data of 7415.3 (monoisotopic molecular weight) and 7420.2 (average molecular weight). The sequencing strategy chosen for Paim I consists of four steps. First, the accurate molecular weights of the intact and tetra-S-carboxymethylated Paim I are determined by fast atom bombardment mass spectrometry. Second, the primary fragments generated by Staphylococcus aureus V8 protease are isolated by reversed-phase high-performance liquid chromatography. The molecular weights of these subpeptides are determined by FABMS. The peptides that must be sequenced are selected by the molecular weights of these subpeptides and the tetra-S-carboxymethylated Paim I. Third, these subpeptides and the whole protein are sequenced by automated Edman degradation. Finally, the primary structure of tetra-S-carboxymethylated Paim I is confirmed by the combination of tryptic, chymotryptic, and S. aureus V8 protease digestion and FABMS. The sequence of Paim I is compared with those of Haim II, Hoe-467A, Z-2685, and AI-3688 because they have different alpha-amylase inhibition spectra against mammalian alpha-amylases but belong to a family of related proteins.     

Microbore HPLC/mass spectrometry for the analysis of peptide mixtures using a continuous flow interface

Microbore HPLC techniques have been combined with fast atom bombardment mass spectrometry to provide HPLC/MS capabilities for the analysis of mixtures of peptides and small proteins. The interface between the liquid chromatograph and mass spectrometer is a continuous flow direct insertion probe which contains a fused silica capillary that delivers the eluting solvent to the FAB source of the mass spectrometer at a rate of 5-10 microL/min. Data are presented for the analysis of several mixtures of peptides ranging in molecular weights from about 900 to 6000 daltons. In addition, the analysis of 100 pmol of a tryptic digest of whale myoglobin is shown where 16 of the possible 19 peptides were identified in the mass range m/z 2200-250. The advantages of this approach to HPLC/MS are a relatively high sensitivity because of the low flow rates and low background, and the ability to detect high molecular weight compounds.     

Mass spectral analysis of murine epidermal growth factor

Fast atom bombardment mass spectrometry has been used to characterize epidermal growth factor isolated from mouse submaxillary glands. The preparation is found to consist of two peptides, one of which has the average molecular weight predicted for the familiar gene product. The molecular weight of the second component is found to be reduced by the mass of one asparagine residue. These observations are discussed in light of previous reports of heterogeneity.     

High-performance size-exclusion chromatography of peptides

Gel filtration on soft gels has been employed for over 40 years for the separation, desalting and molecular weight estimation of peptides and proteins. Technical improvements have given rise to high-performance size-exclusion chromatography (HPSEC) on rigid supports, giving more rapid run times and increased resolution. Initially, these packings were more suitable for the separation of proteins than of peptides, but supports that operate in the fractionation range <10,000 Daltons (Da) are now available. In this report, HPSEC is described in relation to its application to peptides, especially regarding purification, estimation of molecular weight and study of molecular associations.