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.     

Venomics: unravelling the complexity of animal venoms with mass spectrometry

Animal venoms and toxins are now recognized as major sources of bioactive molecules that may be tomorrow's new drug leads. Their complexity and their potential as drug sources have been demonstrated by application of modern analytical technologies, which have revealed venoms to be vast peptide combinatorial libraries. Structural as well as pharmacological diversity is immense, and mass spectrometry is now one of the major investigative tools for the structural investigation of venom components. Recent advances in its use in the study of venom and toxins are reviewed. The application of mass spectrometry techniques to peptide toxin sequence determination by de novo sequencing is discussed in detail, in the light of the search for novel analgesic drugs. We also present the combined application of LC-MALDI separation with mass fingerprinting and ISD fragmentation for the determination of structural and pharmacological classes of peptides in complex spider venoms. This approach now serves as the basis for the full investigation of complex spider venom proteomes, in combination with cDNA analysis.     

Application of MALDI TOF/TOF mass spectrometry and collision-induced dissociation for the identification of disulfide-bonded peptides

This paper describes a method for the fast identification and composition of disulfide-bonded peptides. A unique fragmentation signature of inter-disulfide-bonded peptides is detected using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF)/TOF mass spectrometry and high-energy collision-induced dissociation (CID). This fragmentation pattern identifies peptides with an interconnected disulfide bond and provides information regarding the composition of the peptides involved in the pairing. The distinctive signature produced using CID is a triplet of ions resulting from the cleavage of the disulfide bond to produce dehydroalanine, cysteine or thiocysteine product ions. This method is not applicable to intra-peptide disulfide bonds, as the cleavage mechanism is not the same and a triplet pattern is not observed. This method has been successfully applied to identifying disulfide-bonded peptides in a number of control digestions, as well as study samples where disulfide bond networks were postulated and/or unknown.     

Epitope mapping on bovine prion protein using chemical cross-linking and mass spectrometry

An analytical strategy for the analysis of antigen epitopes by chemical cross-linking and mass spectrometry is demonstrated. The information of antigen peptides involved in the binding to an antibody can be obtained by monitoring the antigen peptides modified by a partially hydrolyzed cross-linker in the absence and in the presence of an antibody. This approach was shown to be efficient for characterization of the epitope on bovine prion protein bPrP(25-241) specifically recognized by a monoclonal antibody, 3E7 (mAb3E7), with only a small amount of sample (200 picomoles) needed. After cross-linking of the specific immuno complex, a matrix-assisted laser desorption/ionization (MALDI) mass spectrometer equipped with an ion conversion dynode (ICD) high-mass detector was used to optimize the amount of cross-linked complex formed at 202 kDa before proteolytic digestion. To identify the cross-linked peptides after proteolysis without ambiguity, isotope-labeled cross-linkers, disuccinimidyl suberate (DSS-d0/d12) and disuccinimidyl glutarate (DSG-d0/d6), together with high-resolution Fourier transform ion-cyclotron resonance mass spectrometry (FTICR-MS) were used. As a result, a complete fading of the peak intensities corresponding to the peptides representing the epitope was observed when bPrP/mAb3E7 complexes were formed.     

Fusaricidins from Paenibacillus polymyxa M‐1, a family of lipohexapeptides of unusual complexity—a mass spectrometric study

Paenibacillus polymyxa are rhizobacteria with a high potential to produce natural compounds of biotechnological and medical interest. Main products of P . polymyxa are fusaricidins, a large family of antifungal lipopeptides with a 15‐guanidino‐3‐hydroxypentadecanoic acid (GHPD) as fatty acid side chain. We use the P . polymyxa strain M‐1 as a model organism for the exploration of the biosynthetic potential of these rhizobacteria. Using matrix‐assisted laser‐desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) about 40 new fusaricidins were detected which were fractionated by reversed‐phase (rp) HPLC. Their structure was determined by MALDI‐LIFT‐TOF/TOF fragment analysis. The dominant fragment in the product ion spectra of fusaricidins appeared at m /z 256.3, 284.3 and 312.4, respectively, indicating variations in their fatty acid part. Two new subfamilies of fusaricidins were introduced which contain guanidino‐3‐hydroxyhepta‐ and nonadecanoic acid as fatty acid constituents. Apparently, the end‐standing guanidine group is not modified as shown by direct infusion nano‐electrospray ionization mass spectrometry (nano‐ESI MS). The results of this study suggest that advanced mass spectrometry is the method of choice for investigating natural compounds of unusual diversity, like fusaricidins. Copyright © 2016 John Wiley & Sons, Ltd.     

Enrichment of N-terminal sulfonated peptides by a water-soluble fullerene derivative and its applications to highly efficient proteomics

Recent studies have shown that N-terminal sulfonation of tryptic peptides by various sulfonating molecules greatly improves their post-source decay processes (e.g., in matrix-assisted laser desorption ionization) or the gas phase fragmentation processes (e.g., in tandem mass spectrometer), enhancing the ability to identify their sequences de novo. In the present work, we have demonstrated that incorporation of water-soluble C₆₀-N,N-dimethylpyrrolidinium iodide selectively precipitates the 4-sulfophenyl isothiocyanate-modified peptide (SPITC-GGYR, SPITC-ASHLGLAR) by forming a noncovalent ion pair to the SO₃⁻ group of the SPITC, and thereby the C₆₀ derivative can be utilized to enrich the modified peptide. Electrospray ionization (ESI) mass analyses show that the cationic SPITC-GGYR and SPITC-ASHLGLAR species are well separated from unmodified peptides and the modified peptides are subsequently detached from the C₆₀ derivative upon using an acidic solution.     

Glycation sites in neoglycoglycoconjugates from the terminal monosaccharide antigen of the O‐PS of Vibrio cholerae O1, serotype Ogawa,and BSA revealed by matrix‐assisted laser desorption–ionization tandem mass spectrometry

We present the MALDI‐TOF/TOF‐MS analyses of various hapten–bovine serum albumin (BSA) neoglycoconjugates obtained by squaric acid chemistry coupling of the spacer‐equipped, terminal monosaccharide of the O‐specific polysaccharide of Vibrio cholerae O1, serotype Ogawa, to BSA. These analyses allowed not only to calculate the molecular masses of the hapten–BSA neoglycoconjugates with different hapten–BSA ratios (4.3, 6.6 and 13.2) but, more importantly, also to localize the covalent linkages (conjugation sites) between the hapten and the carrier protein. Determination of the site of glycation was based on comparison of the MALDI‐TOF/TOF‐MS analysis of the peptides resulting from the digestion of BSA with similar data resulting from the digestion of BSA glycoconjugates, followed by sequencing by MALDI‐TOF/TOF‐MS/MS of the glycated peptides. The product‐ion scans of the protonated molecules were carried out with a MALDI‐TOF/TOF‐MS/MS tandem mass spectrometer equipped with a high‐collision energy cell. The high‐energy collision‐induced dissociation (CID) spectra afforded product ions formed by fragmentation of the carbohydrate hapten and amino acid sequences conjugated with fragments of the carbohydrate hapten. We were able to identify three conjugation sites on lysine residues (Lys235, Lys437 and Lys455). It was shown that these lysine residues are very reactive and bind lysine specific reagents. We presume that these Lys residues belong to those that are considered to be sterically more accessible on the surface of the tridimensional structure. The identification of the y‐series product ions was very useful for the sequencing of various peptides. The series of a‐ and b‐product ions confirmed the sequence of the conjugated peptides. Copyright © 2010 John Wiley & Sons, Ltd.     

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI TOF MS) study of Huperzine A, a natural anti-Alzheimer's disease product, its derivatization and its detection by highly sensitive laser induced fluorescence (LIF)

Huperzine A, a reversible acetylcholinesterase inhibitor for the treatment of Alzheimer disease (HupA), was studied using an (MALDI TOF MS) instrument in MALDI mode. The formation of a HupA dimmer in a vacuum was observed and several matrices were found that were able to inhibit its formation. The structures of the neutral and protonated form of the HupA molecule were calculated and optimized using a Hyperchem program. Detection limit using MALDI TOF MS in the model sample was 5.3 pg. MALDI TOF MS was also applied to the direct detection of the drug in medical preparations and in human serum. The limit of detection in plasma was 14.2 pg with a signal-to-noise ratio of 3:1. However, the sensitivity was not as high as it usually is in MALDI. Therefore, a new method for the derivatization of HupA was developed using fluorescent labelling with rhodamine B isothiocyanate (RBITC). A limit of detection using capillary electrophoresis laser induced fluorescence detection (CE-LIF) equal to 4 × 10⁻⁹ mol l⁻¹ was reached.     

Probing peptide libraries from Conus achatinus using mass spectrometry and cDNA sequencing: identification of delta and omega-conotoxins

The peptide library present in the venom of the piscivorous marine snail Conus achatinus has been probed using a combination of mass spectrometry and cDNA sequencing methods. Matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) analysis, before and following global reduction/alkylation of peptide mixtures, permits the rapid classification of individual components on the basis of the number of disulfide bonds. Mass fingerprinting and the reverse phase HPLC retention times permit a further deconvolution of the library in terms of peptide size and hydrophobicity. Sequencing of cDNA derived using O-superfamily specific primers yielded five complete conotoxin precursor sequences, ranging in polypeptide length from 75-87 residues containing six Cys residues at the C-terminus. Sequence analysis permits classification of the five putative mature peptides (Ac 6.1 to Ac 6.5) as delta, omega, and omega-like conotoxins. The presence of these predicted peptides in crude venom was established by direct matrix assisted laser desorption ionization tandem mass spectrometry (MALDI-MS/MS) sequencing following trypsin digestion of the peptide mixture after global reduction/alkylation. The determination of partial peptide sequences and comparison with the predicted sequences resulted in the identification of four of the five predicted conotoxins. The characterization of posttranslationally modified analogs, which are hydroxylated at proline or amidated at the C-terminus is also demonstrated. Crude venom analysis should prove powerful in studying both inter- and intra-species variation in peptide libraries.     

A novel method for identification and relative quantification of N-terminal peptides using metal-element-chelated tags coupled with mass spectrometry

By means of the reaction between a DOTA-NHS-ester bifunctional reagent and N-terminal peptides of proteins,and then chelation of lanthanide metal ions as tags,we established a novel method for the identification of N-terminal peptides of proteins and their relative quantification using metal-element-chelated tags coupled with mass spectrometry.The experimental results indicate that metal elements are able to completely label N-terminal peptides at the protein level.The N-terminal peptides are enriched as the peptides digested with trypsin are selectively eliminated by isothiocyanate-coupled silica beads.We successfully identified the N-terminal peptides of 158 proteins of Thermoanaerobacter tengcongensis incubated at 55 and 75°C,among which N-terminal peptides of 24 proteins are partially acetylated.Moreover,metal-element tags with high molecule weights make it convenient for N-terminal peptides consisting of less than 6 amino acids to be identified;these make up 55percent of the identified proteins.Finally,we developed a general approach for the relative quantification of proteins based on N-terminal peptides.We adopted lysozyme and ribonuclease B as model proteins;the correlation coefficients(R2)of the standard curves for the quantitative method were 0.9994 and 0.9997,respectively,with each concentration ratio ranging from0.1 to 10 and both relative standard derivations(RSD)measured at less than 5%.In T.tengcongensis at two incubation temperatures,80 proteins possess quantitative information.In addition,compared with the proteins of T.tengcongensis incubated at 55°C,in T.tengcongensis incubated at 75°C,7 proteins upregulate whereas 16 proteins downregulate,and most differential proteins are related to protein synthesis.     

Fragmentation behavior of glycated peptides derived from D-glucose, D-fructose and D-ribose in tandem mass spectrometry

Nonenzymatic glycosylation (or glycation) is a common nonenzymatic side-chain specific sequence-independent posttranslational modification formed by the reaction of reducing carbohydrates with free amino groups. Thus, proteins can react with aldoses or ketoses to yield Amadori or Heynes compounds, respectively. Here, the fragmentation behavior of D-glucose and D-ribose-derived Amadori peptides as well as D-fructose-derived Heynes peptides were studied by collision-induced fragmentation (CID) after electrospray (ESI) or matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). All three sugar moieties displayed characteristic fragmentation patterns accompanying the parent and the fragment ions, which could be explained by consecutive losses of water and formaldehyde. Glucose-derived Amadori parent and fragment ions displayed losses of 18, 36, 54, 72, and 84 u at a characteristic intensity distribution compared with losses of 18, 36, 54, 72, 84, and 96 u for D-fructose-derived ions and losses of 18, 36, and 54 u for ribose-derived ions. Furthermore, each sugar moiety produced indicative lysine-derived immonium ions that were successfully used in a precursor ion scan analysis to identify Amadori peptides in a tryptic digest of bovine serum albumin (BSA) glycated with D-glucose. BSA was modified on lysine residues at positions 36, 160, 235, 256, 401, and 548.     

Characterization and de novo sequencing of snow crab tropomyosin enzymatic peptides by both electrospary ionization and matrix‐assisted laser desorption ionization QqToF tandem mass spectrometry

The protein tropomyosin (TM) is a known major allergen present in shellfish causing frequent food allergies. TM is also an occupational allergen generated in the working environment of snow crab (Chionoecetes opilio) processing plants. The TM protein was purified from both claw and leg meats of snow crab and analyzed by electrospray ionization and matrix‐assisted laser desorption/ionization (MALDI) using hybrid quadruple time‐of‐flight tandem mass spectrometry (QqToF‐MS). The native polypeptide molecular weight of TM was determined to be 32 733 Da. The protein was further characterized using the ‘bottom‐up’ MS approach. A peptide mass fingerprinting was obtained by two different enzymatic digestions and de novo sequencing of the most abundant peptides performed. Any post‐translational modifications were identified by searching their calculated and predicted molecular weights in precursor ion spectra. The immunological reactivity of snow crab extract was evaluated using specific antibodies and allergenic reactivity assessed with serum of allergic patients. Subsequently, a signature peptide for TM was identified and evaluated in terms of identity and homology using the basic local alignment search tool (BLAST). The identification of a signature peptide for the allergen TM using MALDI‐QqToF‐MS will be critical for the sensitive and specific quantification of this highly allergenic protein in the work place. Copyright © 2010 John Wiley & Sons, Ltd.     

Improved detection of phosphopeptides by negative ion matrix-assisted laser desorption/ionization mass spectrometry using a proton sponge co-matrix

Analysis of phosphopeptides is an important task in proteomic studies. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a technique very commonly used for such a purpose. Analysis of phosphopeptides by MALDI-MS is, however, still a challenging task due to the low ionization efficiency of phosphopeptides. In this study, we reported that by using a proton sponge 1,8-bis(dimethyl-amino)naphthalene (DMAN) as a co-matrix, detection of phosphopeptides by negative ion MALDI-MS could be greatly improved. Combination of DMAN with another matrix 6-aza-2-thiothymine (ATT) and additive diammonium hydrogen citrate (DHC) allowed much lower limit of detection, significantly reduced signal suppression effects and improved position-to-position reproducibility for detection of phosphopeptides by negative ion MALDI-MS. Potential applications of the matrix system in qualification of phosphopeptides and analysis of proteolytic digests of phosphorylated proteins were also demonstrated in this study.     

Characterization of peptides by liquid chromatography/electrospray ionization mass spectrometry using silver nitrate as a post-column complexant

Two model peptides, des-Arg1-bradykinin (DAB) and bradykinin (B), were cationized by Ag+ after their separation by reversed-phase liquid chromatography (RPLC) prior to mass spectrometry (MS). Silver nitrate solution was used as a post-column reagent. The RPLC and MS experimental conditions were optimized using flow injection in order to obtain sufficiently abundant silver adducts to permit MS/MS experiments. The use of water-methanol with 0.1% formic acid as mobile phase allowed a good chromatographic separation of the two peptides with a polymeric stationary phase and sufficiently abundant silver-containing adducts, [M + Ag + H]2+ and [M + 2Ag]2+. The gas-phase dissociation of [DAB + Ag + H]2+ and [DAB + 2Ag]2+ led to interpretable mass spectra during the on-line cationization experiment. Most of the ions obtained by dissociating [DAB + Ag + H]2+ and [DAB + 2Ag]2+ species are silver-containing ions but the ions produced depend on the parent. The ions coming from the dissociation of the doubly charged silver adducts [DAB + Ag + H]2+ or [DAB + 2Ag]2+ are of interest compared with those coming from the singly charged silver species or doubly charged protonated species. The fragmentation of the doubly charged silver adducts provides ions over the entire mass range. Although the presence of several prolines in des-Arg1-bradykinin prevents the formation of some expected ions, the observation of triplets [an-H + Ag]+, [bn-H + Ag]+ and [bn + OH + Ag]+ produced by the dissociation of on-line Ag(+)-cationized peptides could contribute to greater success of automatic sequencing of peptides.     

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。     

Improved analysis of membrane protein by PVDF-aided, matrix-assisted laser desorption/ionization mass spectrometry

Characterization of membrane proteins remains an analytical challenge because of difficulties associated with tedious isolation and purification. This study presents the utility of the polyvinylidene difluoride (PVDF) membrane for direct sub-proteome profiling and membrane protein characterization by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The hydrophobic adsorption of protein, particularly membrane proteins, on the PVDF surface enables efficient on-PVDF washing to remove high concentrations of detergents and salts, such as up to 5% sodium dodecyl sulfate (SDS). The enhanced spectrum quality for MALDI detection is particularly notable for high molecular weight proteins. By using on-PVDF washing prior to MALDI detection, we obtained protein profiles of the detergent-containing and detergent-insoluble membrane fractions from Methylococcus capsulatus (Bath). Similar improvements of signal-to-noise ratios were shown on the MALDI spectra for proteins electroblotted from SDS-polyacrylamide gel electrophoresis (SDS-PAGE) onto the PVDF membrane. We have applied this strategy to obtain intact molecular weights of the particulate methane monooxygenase (pMMO) composed of three intrinsic membrane-bound proteins, PmoA, PmoB, and PmoC. Together with peptide sequencing by tandem mass spectrometry, post-translational modifications including N-terminal acetylation of PmoA and PmoC and alternative C-terminal truncation of PmoB were identified. The above results show that PVDF-aided MALDI-MS can be an effective approach for profiling and characterization of membrane proteins.     

Characterization and identification of multiple constituents in Yinhuang granules by high-performance liquid chromatography with diode-array and time-of-flight mass spectrometry detection

A fast high-performance liquid chromatography (HPLC) method with diode-array detection (DAD) and time-of-flight mass spectrometry (TOF/MS) has been developed for the analysis of multi-constituent in Yinhuang granules, a well-known combined herbal remedy prepared from the extract mixtures of Flos Lonicerae and Radix Scutellariae. The fast HPLC analysis was performed on an Agilent ZorBax SB-C(18) column (4.6×50 mm, 1.8 μm) and 0.2% aqueous formic acid and acetonitrile was the optimum mobile phase for gradient elution in 17 min, which is five times faster than the performance of conventional columns packed with 5.0 μm particles. With various fragmentor voltages in TOF/MS, accurate mass measurements (<5 ppm error) for molecular ions and characteristic fragment ions represented reliable identification criteria for different constituents. A total of 28 compounds, including nine phenolic acids, three iridoid glycosides and nine saponins from Flos Lonicerae and seven flavonoids from Radix Scutellariae, were identified or tentatively characterized in the extract of Yinhuang granules. The established fast HPLC-DAD-TOF/MS method turns out to be useful and efficient for quality control of this commonly used Chinese herbal preparation.     

Comprehensive chemical profiling of Guizhi Fuling capsule by the combined use of gas chromatography-mass spectrometry with a deconvolution software and rapid-resolution liquid chromatography quadrupole time-of-flight tandem mass spectrometry

Herbal formulations are complex natural mixtures. Researchers usually tend to focus more on analysis of nonvolatile components but pay less attention to volatile compounds. In this study, an analytical strategy combining two approaches was established for comprehensive analysis of herbal formulations. Guizhi Fuling capsule (GFC), a drug approved by the FDA to enter phase II clinical trial for treatment of primary dysmenorrhea, was taken as a case for analysis. Gas chromatography–mass spectrometry (GC‐MS) with automated mass spectral deconvolution and identification system (AMDIS) led to rapid identification of 48 volatile components including four acetophenones, three fatty acid esters, 13 phenylpropanoids and 19 sesquiterpenes. Most of them were found from Guizhi. The volatile oils of Guizhi have been proved to exhibit many pharmacological activities. This is helpful in understanding the pharmacological mechanism of GFC. Furthermore, AMDIS turned out to be efficient and reliable for analysis of complex herbal formulations. Rapid‐resolution liquid chromatography (RRLC) coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry (ESI‐Q‐TOF MS/MS) allowed the identification of 70 nonvolatile components including six acetophenones, 12 galloyl glucoses, 31 monoterpene glycosides, three phenols and 12 triterpene acids. Fragmentation behaviors of assigned components, especially triterpene acids, which are hard to identify by low‐resolution MS, were first investigated by TOF MS/MS. Characteristic ions and typical loss of assigned triterpene acids were summarized. Combinatorial use of GC‐MS‐AMDIS and RRLC‐ESI‐Q‐TOF MS/MS could be of great help in global qualitative analysis of GFC, as well as other herbal products. Copyright © 2012 John Wiley & Sons, Ltd.     

化学衍生辅助液相色谱-串联质谱测定枇杷膏中的齐墩果酸和熊果酸

建立了一种液相色谱-串联质谱(LC-MS/MS)同时测定齐墩果酸和熊果酸含量的分析方法,利用衍生化试剂N,N-二甲基乙二胺(DMED)和d4-N,N-二甲基乙二胺(d4-DMED)分别衍生样品和标准工作溶液中的目标分析物,并将标准工作液的衍生产物作为稳定同位素内标。该方法线性关系良好,相关系数均0.99;齐墩果酸和熊果酸的检出限(S/N=3)分别为0.92 ng/L和1.06 ng/L,加标回收率分别为98.7%~102.7%和97.2%~105.0%。该方法简单、快速、准确,可满足枇杷膏中齐墩果酸和熊果酸高灵敏度检测的需求。     

Improvements in protein identification confidence and proteome coverage for human liver proteome study by coupling a parallel mass spectrometry/mass spectrometry analysis with multi-dimensional chromatography separation

Recently, matrix-assisted laser desorption ionization (MALDI) technique has been shown to be complementary to electrospray ionization (ESI) with respect to the population of peptides and proteins that can be detected. In this study, we tried to hyphenate MALDI-TOF-TOF-MS and ESI-QUADRUPOLE-TOF-MS with a single 2D liquid chromatography for complicated protein sample analysis. The effluents of RPLC were split into two parts for the parallel MS/MS detection. After optimizing the operation conditions in LC separation and MS identification, a total of 1149 proteins were identified from the global lysate of normal human liver (NHL) tissue. Compared to the single MS/MS detection, the combined analysis increased the number of proteins identified (more than 25%) and enhanced the protein identification confidence. Proteins identified were categorized and analyzed based upon their cellular location, biological process and molecular function. The identification results demonstrated the application potential of a parallel MS/MS analysis coupled with multi-dimensional LC separation for complicated protein sample identification, especially for proteome analysis, such as human tissues or cells extracts.