Determination of N-linked glycosylation of yeast external invertase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The extent of N-glycosylation of yeast external invertase at each of the 14 potential sites was determined by the combination of proteolytic digestions and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS). The average molecular mass of the intact external invertase was determined as 97 kDa by MALDI/TOF-MS. The intact protein was digested with trypsin, Lys-C and Asp-N, followed by high-performance liquid chromatographic separation. The proteolytic digests were analyzed by MALDI/MS screening for the glycopeptides. The glycopeptides were then treated with peptide:N-glycosidase F (PNGase F) and/or endo-beta-N-acetylglucosaminidase (Endo H) and the molecular mass of the deglycosylated peptide was determined by MALDI/MS and matched with the peptide predicted by a computer program. The sequences of some peptides or deglycosylated peptides were identified by the MALDI post-source decay technique. The size of the oligosaccharide, the degree of glycosylation and the distribution of the oligosaccharides at each individual potential glycosylation site were characterized. This information goes for beyond previously published data and sometimes differs from them. During this study, the amino acid sequence originally derived from the DNA sequence of the gene coding for invertase was also verified and it was found that this protein when expressed from SUC2 gene might be created as more than one sequence which differ by a few amino acid substitutions (Asn58<-->Thr, Asn65-->His and Val412<-->Ala).     

Microwave-assisted acid hydrolysis of proteins combined with liquid chromatography MALDI MS/MS for protein identification

Simple and efficient digestion of proteins, particularly hydrophobic membrane proteins, is of significance for comprehensive proteome analysis using the bottom-up approach. We report a microwave-assisted acid hydrolysis (MAAH) method for rapid protein degradation for peptide mass mapping and tandem mass spectrometric analysis of peptides for protein identification. It uses 25% trifluoroacetic acid (TFA) aqueous solution to dissolve or suspend proteins, followed by microwave irradiation for 10 min. This detergent-free method generates peptide mixtures that can be directly analyzed by liquid chromatography (LC) matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) without the need of extensive sample cleanup. LC-MALDI MS/MS analysis of the hydrolysate from 5 microg of a model transmembrane protein, bacteriorhodopsin, resulted in almost complete sequence coverage by the peptides detected, including the identification of two posttranslational modification sites. Cleavage of peptide bonds inside all seven transmembrane domains took place, generating peptides of sizes amenable to MS/MS to determine possible sequence errors or modifications within these domains. Cleavage specificity, such as glycine residue cleavage, was observed. Terminal peptides were found to be present in relatively high abundance in the hydrolysate, particularly when low concentrations of proteins were used for MAAH. It was shown that these peptides could still be detected from MAAH of bacteriorhodopsin at a protein concentration of 1 ng/microl or 37 fmol/microl. To evaluate the general applicability of this method, it was applied to identify proteins from a membrane protein enriched fraction of cell lysates of human breast cancer cell line MCF7. With one-dimensional LC-MALDI MS/MS, a total of 119 proteins, including 41 membrane-associated or membrane proteins containing one to 12 transmembrane domains, were identified by MS/MS database searching based on matches of at least two peptides to a protein.     

Characterization of implant device materials using size-exclusion chromatography with mass spectrometry and with triple detection

A more complete understanding of the raw materials used for making implant device materials becomes increasingly important in the medical device industry. Often such detailed information requires utilization of a combination of analytical techniques. In this work, we characterize a poly(dimethyl siloxane) (PDMS) material using on-line size exclusion chromatography (SEC) with electrospray ionization (ESI) and matrix assisted laser desorption ionization (MALDI) mass spectrometry (MS) techniques. Here, we obtain detailed molecular compositional information such as repeat units, end group chemistry, and identification of impurities in both the high and low mass range. SEC with light scattering, viscosity, and refractive index detection (triple detection) is used to obtain information on a small quantity of high mass impurity that was undetected by both SEC-ESI and MALDI MS techniques. SEC with triple detection measures absolute molecular weights and molecular weight distributions. We compare average molecular weight values of the implantable device polymer obtained by SEC with triple detection, SEC-ESI, and SEC-MALDI MS techniques.     

Quantification of Greenland halibut serum vitellogenin: a trip from the deep sea to the mass spectrometer

This paper focuses on the sequential steps involved in developing a technique for quantifying Greenland halibut vitellogenin, a serum protein biomarker, using a comprehensive mass spectrometric approach. In the first phase of this study, in‐gel trypsin digestions of serum proteins separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) were analyzed by matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS). A characteristic band around a molecular mass of 185 kDa, present in the mature female specimens, but absent in the male samples, was identified as vitellognin according to the peptide mass fingerprint obtained by MALDI‐MS. Subsequently, MALDI and electrospray ionization tandem mass spectrometry (ESI‐MS/MS) analyses were performed on the digest of the vitellogenin band for de novo sequencing. From these studies, a characteristic 'signature' peptide (sequence: FFGQEIAFANIDK) was selected from a list of candidate peptides as a surrogate analytical standard used for quantification purposes. Sample preparation for vitellogenin quantification consisted of a simple one‐step overnight trypsin digestion. Samples were spiked with an isotopologue signature peptide standard and analyzed by high‐performance liquid chromatography (HPLC) coupled in‐line to an electrospray quadrupole‐hexapole‐quadrupole tandem mass spectrometer, operated in selective reaction monitoring mode. Transitions [(m/z 750.0 → 1020.4 and 750.0 → 1205.4) and (754.8 → 1028.6 and 754.8 → 1213.2)] were monitored for the signature peptide and the internal standard, respectively. Samples obtained from the field showed that vitellogenin levels were in accordance with fish maturity determined by macroscopic examination of the gonad, proving this technique suitable for measuring vitellogenin as a serum protein biomarker for reproductive maturity in female fish. Copyright © 2009 John Wiley & Sons, Ltd.     

Comparision of FAB and MALDI Mass Spectrometry of Ginsenosides

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蛋白质分子量测定过程中的酸效应

在基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）和电喷雾质谱（ESI-MS）测定蛋白质分子分子量的过程中,适当提高样品的酸度,可提高分析测试的灵敏度。在选定最佳样品分子浓度的基础上,通过适当加入三氟乙酸（TFA）来调整测试样品的酸度,准确测定了标准蛋白质-溶菌酶（lysozyme）的分子量,并且对蛋白质分子在“软电离”质谱中,受酸效应的影响进行了初步探讨。     

Applications of mass spectrometry to food proteins and peptides

The application of mass spectrometry (MS) to large biomolecules has been revolutionized in the past decade with the development of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) techniques. ESI and MALDI permit solvent evaporation and sublimation of large biomolecules into the gaseous phase, respectively. The coupling of ESI or MALDI to an appropriate mass spectrometer has allowed the determination of accurate molecular mass and the detection of chemical modification at high sensitivity (picomole to femtomole). The interface of mass spectrometry hardware with computers and new extended mass spectrometric methods has resulted in the use of MS for protein sequencing, post-translational modifications, protein conformations (native, denatured, folding intermediates), protein folding/unfolding, and protein-protein or protein-ligand interactions. In this review, applications of MS, particularly ESI-MS and MALDI time-of-flight MS, to food proteins and peptides are described.     

Matrix-assisted ionization vacuum for protein detection,fragmentation and PTM analysis on a high resolution linear ion trap-orbitrap platform

Matrix-assisted ionization vacuum (MAIV) is a novel ionization technique that generates multiply charged ions in vacuum without the use of laser ablation or high voltage. MAIV can be achieved in intermediate-vacuum and high-vacuum matrix-assisted laser desorption/ionization (MALDI) sources and electrospray ionization (ESI) sources without instrument modification. Herein, we adapt MAIV onto the MALDI-LTQ-Orbitrap XL platform for biomolecule analysis. As an attractive alternative to MALDI for in solution and in situ analysis of biomolecules, MAIV coupling to high resolution and accurate mass (HRAM) MS instrument has successfully expanded the mass detection range and improved the fragmentation efficiency due to the generation of multiply charged ions. Additionally, the softness of MAIV enables potential application in labile post-translational modification (PTM) analysis. In this study, proteins as large as 18.7 kDa were detected with up to 18 charges; intact peptides with labile PTM were well preserved during the ionization process and characterized MS/MS; peptides and proteins in complex tissue samples were detected and identified both in liquid extracts and in situ. Moreover, we demonstrated that this method facilitates MS/MS analysis with improved fragmentation efficiency compared to MALDI-MS/MS.     

Application of electrospray mass spectrometry and matrix-assisted laser desorption ionization time-of-flight mass spectrometry for molecular weight assignment of peptides in complex mixtures

Electrospray mass spectrometry (ES/MS) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF/MS) were used to provide mass spectra from seven elapid snake venoms. Spectral interpretation was much simpler for MALDI/TOF/MS. ES/MS proved more useful for the provision of molecular weight data for very closely related peptides, but suppression of higher molecular weight compounds was seen to occur during flow injection analysis. MALDI/TOF/MS proved useful for providing a complete picture of the venom, but the low resolution led to obscuring of major ions, and the mass accuracy was poorer for known peptides. Suppression also occurred during MALDI/TOF/MS but could be overcome using alternative matrices because the spectra were very dependent on the choice of matrix. ES/MS and MALDI/TOF/MS provide complementary and confirmatory information such that for the anal sis of complex peptide mixtures (snake venoms), the use of both techniques is desirable.     

Molecular mass determination of plasma-derived glycoproteins by ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with internal calibration

Human plasma-derived antithrombin III (AT-III), factor IX (FIX) and vitronectin (VN) were characterized as native glycoproteins and in their de-N-glycosylated form by means of MALDI mass spectrometry. The average molecular masses of the three complex glycoproteins were determined applying internal calibration with high-mass, well-defined protein calibrants. Internal calibration generated for the 47 kDa yeast protein enolase a mass precision in the continuous and delayed extraction mode of +/-0.12 and +/-0.022%, respectively. The achievable mass accuracy for such a high-mass, unmodified protein was in the range of 0.02% in the continuous mode, which turned out to be better than in the delayed extraction mode. Purification of all (glyco) proteins (even the calibration proteins) by means of ZipTip technology and direct elution with a solvent system containing the appropriate MALDI matrix turned out to be a prerequisite to measure the exact molecular masses with an internal calibration. The average molecular masses of the two different forms of AT-III, namely AT-III(alpha) and AT-III(beta), were shown to be 57.26 and 55.04 kDa, respectively. The 2.22 kDa mass difference is attributed to the known difference in carbohydrate content at one specific site (Asn-135). After exhaustive de-N-glycosylation (by means of PNGase F) of the alpha- and beta-form and subsequent MALDI-MS analysis, average molecular masses of 48.96 and 48.97 kDa, respectively, were obtained. These values are in good agreement (-0.15%) with the calculated molecular mass (49.039 kDa) of the protein part based on SwissProt data. The molecular mass of the heavily post-translational modified glycoprotein FIX was found to be 53.75 kDa with a peak width at 10% peak height of 4.5 kDa, because of the presence of many different posttranslational modifications (N- and O-glycosylation at multiple sites, sulfation, phosphorylation, hydroxylation and numerous gamma-carboxyglutamic acids). MALDI-MS molecular mass determination of the native, size-exclusion chromatography-purified, VN sample revealed that the glycoprotein was present as dimer with molecular mass of 117.74 kDa, which could be corroborated by non-reducing SDS-PAGE. After sample treatment with guanidine hydrochloride and mass spectrometric analysis, a single, new main component was detected. The molecular mass turned out to be 59.45 kDa, representing the monomeric form of VN, known as V75. The determined molecular mass value was shown to be on one hand lower than from SDS-PAGE and on the other higher than the calculated amino acid sequence molecular mass (52 277 Da), pointing to the well-known SDS-PAGE bias and to considerable post-translational modifications. Further treatment of the sample with a reducing agent and subsequent MALDI-MS revealed two new components with molecular masses of 49.85 and 9.41 kDa, corresponding to V65 and V10 subunits of VN. PNGase F digest of the V75 and V65 units and MS analysis, exhibiting a molecular mass reduction of 6.37 kDa in both cases, verified the presence of a considerable amount of N-glycans.     

Metabolite imaging using matrix-enhanced surface-assisted laser desorption/ionization mass spectrometry (ME-SALDI-MS)

We describe here the use of a hybrid ionization approach, matrix-enhanced surface-assisted laser desorption/ionization mass spectrometry (ME-SALDI-MS) in bioimaging. ME-SALDI combines the strengths of traditional matrix-assisted laser desorption/ionization (MALDI) and SALDI and enables successful MS imaging of low-mass species with improved detection sensitivity. Using 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) as the MS standard, MS performances of MALDI, SALDI, and ME-SALDI are systematically compared. The analyte desorption and ionization mechanism in ME-SALDI is qualitatively speculated based on the observation of significantly reduced matrix background and improved survival yields of molecular ions. Improvements in detection sensitivity of low-mass species using ME-SALDI over MALDI in imaging are demonstrated with mouse heart and brain tissues.     

Properties of mouse vomeronasal receptor and assessment of its role in pheromone signalling

Vomeronasal type 2 receptor (V2Rx) from Swiss mouse (Mus musculus (L.)) was analyzed by high-resolution ion-exchange chromatography, reversed-phase high-performance liquid chromatography (RP-HPLC), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), Ion Spray tandem mass spectrometry (MS/MS), 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and 1-aminoanthracene (1-AMA) fluorometric assay. Vomeronasal sensory neuronal cell bound proteins were resolved into major protein peaks. Several proteins were identified and subsequently purified as the V2Rx receptor on 10% SDS-PAGE with trace amounts of other protein bands. The molecular weight of the identified V2Rx was 109 kDa. MALDI-TOF and micro-sequencing experiments demonstrated that the identified V2Rx receptor shared considerable sequence similarity with vomeronasal receptor type 2 (NCBI Accession Number AB267725), which is a seven transmembrane peptide with 912 amino acid residues. The molecular characterization revealed that the N-terminus of the V2Rx receptor contained the 11GAEAAE16 domain involved in pheromone signalling. The biometric assay (octanamine-V2Rx binding) showed the identified V2Rx receptor and mouse sex pheromone to 2-octanamine (methyl heptyl) in a 1:1 ratio. Uptake of odourants determined in physiological condition showed enhanced V2Rx receptors as volatile hydrophobic pheromone receptors in the vomeronasal neuron of the Swiss mouse.     

Electrospray-assisted laser desorption/ionization and tandem mass spectrometry of peptides and proteins

We have constructed an electrospray-assisted laser desorption/ionization (ELDI) source which utilizes a nitrogen laser pulse to desorb intact molecules from matrix-containing sample solution droplets, followed by electrospray ionization (ESI) post-ionization. The ELDI source is coupled to a quadrupole ion trap mass spectrometer and allows sampling under ambient conditions. Preliminary data showed that ELDI produces ESI-like multiply charged peptides and proteins up to 29 kDa carbonic anhydrase and 66 kDa bovine albumin from single-protein solutions, as well as from complex digest mixtures. The generated multiply charged polypeptides enable efficient tandem mass spectrometric (MS/MS)-based peptide sequencing. ELDI-MS/MS of protein digests and small intact proteins was performed both by collisionally activated dissociation (CAD) and by nozzle-skimmer dissociation (NSD). ELDI-MS/MS may be a useful tool for protein sequencing analysis and top-down proteomics study, and may complement matrix-assisted laser desorption/ionization (MALDI)-based measurements.     

Phosphoric acid enhances the performance of Fe(III) affinity chromatography and matrix-assisted laser desorption/ionization tandem mass spectrometry for recovery, detection and sequencing of phosphopeptides

An integrated analytical strategy for enrichment, detection and sequencing of phosphorylated peptides by matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry (MS/MS) is reported. o-Phosphoric acid was found to enhance phosphopeptide ion signals in MALDI-MS when used as the acid dopant in 2,5-dihydroxybenzoic acid (2,5-DHB) matrix. The effect was largest for multiply phosphorylated peptides, which exhibited an up to ten-fold increase in ion intensity as compared with standard sample preparation methods. The enhanced phosphopeptide response was observed during MALDI-MS analysis of several peptide mixtures derived by proteolytic digestion of phosphoproteins. Furthermore, the mixture of 2,5-DHB and o-phosphoric acid was an excellent eluant for immobilized metal affinity chromatography (IMAC). Singly and multiply phosphorylated peptide species were efficiently recovered from Fe(III)-IMAC columns, reducing sample handling for phosphopeptide mapping by MALDI-MS and subsequent phosphopeptide sequencing by MALDI-MS/MS. The enhanced response of phosphopeptide ions in MALDI facilitates MS/MS of large (>3 kDa) multiply phosphorylated peptide species and reduces the amount of analyte needed for complete characterization of phosphoproteins.     

Phosphopeptide quantitation using amine-reactive isobaric tagging reagents and tandem mass spectrometry: application to proteins isolated by gel electrophoresis

Polyacrylamide gel electrophoresis is widely used for protein separation and it is frequently the final step in protein purification in biochemistry and proteomics. Using a commercially available amine-reactive isobaric tagging reagent (iTRAQ) and mass spectrometry we obtained reproducible, quantitative data from peptides derived by tryptic in-gel digestion of proteins and phosphoproteins. The protocol combines optimized reaction conditions, miniaturized peptide handling techniques and tandem mass spectrometry to quantify low- to sub-picomole amounts of (phospho)proteins that were isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Immobilized metal affinity chromatography (FeIII-IMAC) was efficient for removal of excess reagents and for enrichment of derivatized phosphopeptides prior to matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis. Phosphopeptide abundance was determined by liquid chromatography/tandem mass (LC/MS/MS) using either MALDI time-of-flight/time-of-flight (TOF/TOF) MS/MS or electrospray ionization quadrupole time-of-flight (ESI-QTOF) MS/MS instruments. Chemically labeled isobaric phosphopeptides, differing only by the position of the phosphate group, were distinguished and characterized by LC/MS/MS based on their LC elution profile and distinct MS/MS spectra. We expect this quantitative mass spectrometry method to be suitable for systematic, comparative analysis of molecular variants of proteins isolated by gel electrophoresis.     

Comparative studies of poly(dimethyl siloxanes) using automated GPC-MALDI-TOF MS and on-line GPC-ESI-TOF MS

In this study we compare on-line gel permeation chromatography (GPC) electrospray ionization (ESI) time-of-flight (TOF) mass spectrometry (MS) to automated GPC matrix assisted laser desorption ionization (MALDI) TOF MS for poly (dimethylsiloxane) (PDMS) analysis. Average mass values for a hydroxyl-terminated PDMS (OH-PDMS) sample were obtained and compared to traditional GPC that was calibrated with narrow polystyrene standards, by direct ESI and MALDI MS analysis, by a summation of mass spectra of all GPC fractions, and also by the recalibration method determined by both mass spectrometric methods. Quantitatively, the difference noted here between these hyphenated techniques is that GPC-ESI-TOF MS effectively reports the low-mass oligomers and underestimates the high-mass oligomers, while GPC-MALDI-TOF MS effectively reports the high-mass oligomers and underestimates the low-mass oligomers. In the GPC-ESI-TOF MS experiments, ion current suppression was observed in the high molecular weight region. The suppression effect was confirmed by repeatable sample runs and by injecting different PDMS samples. Higher chromatographic resolution was observed for GPC-ESI-TOF MS compared to GPC-MALDI-TOF MS. In fact, truly mono-disperse oligomers were observed in the low molecular weight range from GPC-ESI MS experiments.     

The fragmentation of ethoxylated surfactants by AP-MALDI-QIT

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has become an important technique to characterize the chemical structure of industrial polymer materials. MALDI methods have been developed to address a broad variety of different polymer materials containing different chemistries. One of the key aspects of the typical MALDI experiment is the generation of intact ions. The development of Atmospheric Pressure (AP) MALDI quadrupole ion trap (QIT) instruments has opened another channel to obtaining MS/MS experiments for polymer samples. These experiments provide a new method to obtain chemical structure information from MALDI experiments. Collision-Induced Dissociation (CID) provides an improved MALDI MS/MS experiment that can be done on readily available mass spectrometers. AP MALDI QIT techniques have been successfully applied to a variety of synthetic polymers. This work explores the applicability of AP MALDI QIT methods to relatively low molecular weight ethoxylated surfactants. In these experiments we show the CID fragmentation mass spectra on some ethoxylated surfactants, and demonstrate the existence of analyte matrix clusters.     

Analysis of the saliva from patients with oral cancer by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), this study analyzed the saliva obtained from patients with oral cancer and compared these mass spectra with those obtained from healthy controls. Saliva without pre-treatment was mixed directly with a sinapinic acid matrix. Alpha-amylase (57 kDa) dominated the high mass range in the MALDI mass spectra of the saliva from healthy subjects, but the peak was suppressed for patients with oral cancer and was replaced by a peak at m/z 66 k in the spectra of patients' samples (15 out of 20). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with in-gel tryptic digestion combined with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) was employed to characterize this 66-kDa protein, which was thus shown to be albumin. However, based on SDS-PAGE results, concentrations of both alpha-amylase and albumin in patients' saliva were significantly higher than those in healthy subjects. This discrepancy was shown to be due to MALDI suppression effects due to the albumin. MALDI-MS thus has potential as a possible rapid diagnostic screening tool for oral cancer.     

长白山白眉蝮蛇蛇毒精氨酸酯酶1的研究Ⅰ.纯化、分子量测定及纯度鉴定

利用离子交换 (DEAESephadexA - 50 )和凝胶过滤层析 (SephadexG - 75)技术首次从长白山白眉蝮蛇蛇毒 (AgkistrodonblomhoffiiUssurensis)中纯化得到了一种精氨酸酯酶纯品。经SDS -聚丙烯酰胺凝胶电泳(SDS -PAGE)以及基质辅助激光解吸电离飞行时间质谱 (MALDI/TOF/MS)鉴定为单一纯蛋白 ,分子量为2 991 8.5± 1 5Da ,为进一步研究其结构与功能提供了可靠的依据。     

Automated assignment of ionization states in broad‐mass matrix‐assisted laser desorption/ionization spectra of protein mixtures

A computational technique is presented for the automated assignment of the multiple charge and multimer states (ionization states) in the time‐of‐flight (TOF) domain for matrix‐assisted laser desorption/ionization (MALDI) spectra. Examples of the application of this technique include an improved, automatic calibration over the 2 to 70 kDa mass range and a reduced data redundancy after reconstruction of the molecular spectrum of only singly charged monomers. This method builds on our previously reported enhancement of broad‐mass signal detection, and includes two steps: (1) an automated correction of the instrumental acquisition initial time delay, and (2) a recursive TOF detection of multiple charge states and singly charged multimers of molecular [MH]⁺ ions over the entire record range, based on MALDI methods. The technique is tested using calibration mixtures and pooled serum quality control samples acquired along with clinical study data. The described automated procedure improves the analysis and dimension reduction of MS data for comparative proteomics applications. Copyright © 2009 John Wiley & Sons, Ltd.