Method optimisation for peptide profiling of microdissected breast carcinoma tissue by matrix-assisted laser desorption/ionisation-time of flight and matrix-assisted laser desorption/ionisation-time of flight/time of flight-mass spectrometry

Appropriate methods for the analysis of microdissected solid tumour tissues by matrix-assisted laser desorption/ionisation-time of flight-mass spectrometry (MALDI-TOF MS) are not yet well established. Optimisation of sample preparation was performed first on undissected tissue slices, representing approximately 200 000 cells, which were solubilised either in urea containing buffer, trifluoroethanol/NH4HCO3, 0.1% sodium dodecyl sulphate (SDS) or in 0.1% RapiGest solution, then trypsin digested and analysed by MALDI-TOF MS. Solubilisation in 0.1% SDS resulted in detection of the highest number of sample specific peak signals. Interestingly, there was little overlap in detectable peaks using the different buffers, implying that they can be used complementarily to each other. Additionally, we fractionated tryptic digests on a monolithic high-performance liquid chromatography column. Fractionation of tryptic digest from whole tissue sections resulted in a four-fold increase in the total number of peaks detected. To prove this principle, we used 0.1% SDS to generate peptide patterns from 2000 microdissected tumour and stromal cells from five different breast carcinoma tumours. The tumour and stroma specific peaks could be detected upon comparison of the peptide profiles. Identification of differentially expressed peaks by MALDI-TOF/TOF MS was performed on fractionated tryptic digests derived from a whole tissue slice. In conclusion, we describe a method that is suitable for direct peptide profiling on small amounts of microdissected cells obtained from breast cancer tissues.     

Proteome analysis of Arabidopsis thaliana by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry

In the present study we show results of a large-scale proteome analysis of the recently sequenced plant Arabidopsis thaliana. On the basis of a previously published sequential protein extraction protocol, we prepared protein extracts from eight different A. thaliana tissues (primary leaf, leaf, stem, silique, seedling, seed, root, and inflorescence) and analysed these by two-dimensional gel electrophoresis. A total of 6000 protein spots, from three of these tissues, namely primary leaf, silique and seedling, were excised and the contained proteins were analysed by matrix assisted laser desorption/ionisation time of flight mass spectrometry peptide mass fingerprinting. This resulted in the identification of the proteins contained in 2943 spots, which were found to be products of 663 different genes. In this report we present and discuss the methodological and biological results of our plant proteome analysis.     

Peptide mass fingerprinting by matrix-assisted laser desorption ionization mass spectrometry of proteins detected by immunostaining on nitrocellulose

We have developed an approach that allows peptide mass mapping by matrix-assisted laser desorption ionization-mass spectrometry of proteins visualized on a nitrocellulose membrane by immunochemical detection. Proteins are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), electroblotted onto a nitrocellulose membrane and after blocking with a nonprotein-containing polymer such as polyvinylpyrrolidone 40 (PVP-40) or Tween 20, the proteins are stained with fount India ink. After incubation with primary and, if required, secondary peroxidase-coupled antibodies, immunochemically reactive proteins can be visualized using conventional enhanced chemiluminescence detection and assigned to the India ink-stained membrane by simple superposition. The proteins of interest are excised, submitted to "on-membrane" cleavage and the peptides are analyzed by mass spectrometry. Protein-based blocking reagents normally used in standard immunodetection protocols, such as skimmed milk, can be employed. We have obtained high-quality mass spectra of bovine serum albumin (BSA) detected on an immunoblot with an estimated amount of 100 fmol applied onto the gel, indicating the sensitivity of the present method. In addition, the approach is demonstrated with two other commercially available proteins, a serum protein, the successful identification of a tyrosine phosphorylated protein from total rat liver homogenate and serine phosphorylated proteins from an EcR 293 nuclear extract separated by two-dimensional (2-D) SDS-PAGE.     

Peptide analysis: solid phase extraction-elution on diamond combined with atmospheric pressure matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometry

Electrospray ionization (ESI) has been an indispensable ion generation technique for mass spectrometric analysis of biopolymers such as intact proteins and protein digests operated at atmospheric pressure. Since its advent in 1998, atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) quickly became a popular alternative for the analysis of peptides. Although AP-MALDI sources typically share the same vacuum interface and ion transmission hardware with ESI, it is generally found that ESI is superior in detection sensitivity. Here we present a method based on solid phase extraction and elution with surface-functionalized diamond nanocrystals (which we previously referred to as "SPEED") that not only streamlines AP-MALDI mass spectrometric analyses of peptides and other small biomolecules under typical operational conditions but also outruns ESI in ultimate detectable concentration by at least one order of magnitude.     

Characterisation of bacteria by matrix-assisted laser desorption/ionisation and electrospray mass spectrometry

Chemical analysis for the characterisation of micro-organisms is rapidly evolving, after the recent advent of new ionisation methods in mass spectrometry (MS): electrospray (ES) and matrix-assisted laser desorption/ionisation (MALDI). These methods allow quick characterisation of micro-organisms, either directly or after minimum sample preparation. This review provides a brief introduction to ES and MALDI MS and a discussion of micro-organism characterisation capabilities. Some attention is devoted to the analysis of mixtures of proteins, lipids and other compounds, to the combination of polymerase chain reaction technology and MS, and to the analysis of whole bacteria and their lysates. The review of results produced hitherto is concluded with an outlook on future developments.     

Determination of prolidase activity using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Proline-containing peptides of the X-proline type are cleaved by the dipeptidase prolidase. The classical method of prolidase assay relied on the colorimetric estimation of the liberated proline with ninhydrin using acidic media and heat. This method, however, gave inconsistent results due to the nonspecificity of the ninhydrin color reaction. We report here a method for the detection of the liberated proline using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Human sera were incubated with a mixture containing the dipeptide glycyl-proline in Tris-HCl supplemented with manganese at 37 degrees C for 24h. The samples were precipitated with trifluoroacetic acid and centrifuged. An aliquot of the supernatant was mixed with an equal volume of ferulic acid solution. An aliquot from this mixture was spotted on a stainless steel mass spectrometry grid and analyzed using MALDI-TOF mass spectrometry. The activity of the enzyme was determined by the complete disappearance of the glycyl-proline peak with the concomitant appearance of the proline peak and can be expressed in terms of the ratio of the area beneath the proline to the area beneath the glycyl-proline peak. Subjects homozygous for prolidase deficiency had a ratio ranging from 0.006 to 0.04 while obligatory heterozygotes had a ratio ranging from around 1.1 to 2.4. Normal subjects had ratios ranging from 9 to 239. Using this method we have unambiguously identified subjects with homozygous or heterozygous prolidase deficiency. In addition to the advantage of rapid sample preparation time, this method is highly specific, reproducible, and sensitive.     

Glycoproteomics of N-glycosylation by in-gel deglycosylation and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry mapping: application to congenital disorders of glycosylation

A general strategy for the structural evaluation of N-glycosylation, a common post-translational protein modification, is presented. The methods for the release of N-linked glycans from the gel-separated proteins, their isolation, purification and matrix-assisted laser desorption/ionisation-mass spectrometry (MALDI-MS) analysis of their mixtures were optimised. Since many glycoproteins are available only at low quantities from sodium dodecyl sulphate-polyacrylamide gel electrophoresis or two-dimensional gels, high attention was paid to obtain N-glycan mixtures representing their actual composition in human plasma by in-gel deglycosylation. The relative sensitivity of solid MALDI matrices for MS analysis of acidic N-glycans was compared. The most favourable results for native acidic N-glycans were obtained with 2,4,6-trihydroxyacetophenone monohydrate/diammoniumcitrate as a matrix. This matrix provided good results for both neutral and acidic mixtures as well as for methylated N-glycans. In the second part of this paper the potential of such an optimised MS strategy alone or in combination with high pH anion-exchange chromatography profiling for the clinical diagnosis of congenital disorders of glycosylation is presented.     

Quantification of angiotensin-converting-enzyme-mediated degradation of human chemerin 145-154 in plasma by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry

Chemerin is a chemoattractive protein acting as a ligand for the G-protein-coupled receptor ChemR23/CMKLR1 and plays an important role in the innate and adaptive immunity. Proteolytic processing of its C terminus is essential for receptor binding and physiological activity. Therefore, we investigated the plasma stability of the decapeptide chemerin 145-154 (P(145)-F(154)) corresponding to the C terminus of the physiologically active chemerin variant E(21)-F(154) from human hemofiltrate. For monitoring concentration-time profiles and degradation products we developed a novel matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry procedure using an internal peptide standard (hemorphin LVV-H7) for quantification. The linear range covers 2.5 orders of magnitude in the lower micromolar concentration range (lower limit of quantification 0.312 microg/ml, 0.25 microM) characterized by satisfactory reproducibility (CV < or =9%), accuracy (< or =10%), ruggedness, and recovery (98%). We found that chemerin 145-154 is C-terminally truncated in human citrate plasma by the cleavage of the penultimate dipeptidyl residue. N-terminal truncation was not observed. In contrast to citrate plasma, no degradation was detected in ethylenediammetetraacetate (EDTA) plasma. We identified angiotensin-converting-enzyme (ACE) to be responsible for C-terminal truncation, which could be completely inhibited by EDTA and captopril. These results are relevant to clarify the natural processing of chemerin and the potential involvement of ACE in mediating the immune response.     

Characterization of Cryptosporidium parvum by matrix-assisted laser desorption ionization-time of flight mass spectrometry

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was used to investigate whole and freeze-thawed Cryptosporidium parvum oocysts. Whole oocysts revealed some mass spectral features. Reproducible patterns of spectral markers and increased sensitivity were obtained after the oocysts were lysed with a freeze-thaw procedure. Spectral-marker patterns for C. parvum were distinguishable from those obtained for Cryptosporidium muris. One spectral marker appears specific for the genus, while others appear specific at the species level. Three different C. parvum lots were investigated, and similar spectral markers were observed in each. Disinfection of the oocysts reduced and/or eliminated the patterns of spectral markers.     

基质辅助激光解吸电离飞行时间质谱对阪崎肠杆菌的鉴定

目的 利用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)法对阪崎肠杆菌进行鉴定,建立一种高效检测阪崎肠杆菌的方法,并为该技术的推广使用及阪崎肠杆菌的进一步研究提供科学依据.方法 用MALDI-TOF-MS法检测38株野生阪崎肠杆菌、2株标准菌株和1株阴沟肠杆菌,结果与常规生化鉴定结果对比;同时对在不同培养基上培养的阪崎肠杆菌进行质谱分析比较,对比不同培养基对质谱结果是否有影响;对38株野生菌株质谱图进行聚类分析.结果 38株菌株鉴定结果均为阪崎肠杆菌,与生化鉴定结果一致,且质谱鉴定分值大多在2.0以上.通过MALDI-TOF-MS鉴定方法可以很明显地将阴沟肠杆菌与阪崎肠杆菌两种菌分开.4种培养基对MALDI-TOF-MS鉴定结果的影响不是很明显,TSA比较适合作为阪崎肠杆菌MALDI-TOF-MS鉴定的培养基.通过质谱图谱和离子峰值比较得出,所有菌株在5745 m/z附近均出现高的离子峰,在2871、4740、8288、6260和9488 m/z附近出现离子峰的实验菌株达95％以上;在差异水平在0.5时,MALDI-TOF-MS的聚类分析结果可将所有实验菌株分成5个类型,结合菌株对应的来源和种类分析表明本研究所用菌株与来源和种类之间并无明显关系.结论 MALDI-TOF-MS方法具有准确且精确鉴定阪崎肠杆菌的能力;离子峰5745m/z具有作为阪崎肠杆菌的标记性离子峰的可能;差异水平为0.5进行MALDI-TOF-MS聚类分析,未发现5个类型与来源等具有一定关系,需要进一步研究.     

Analysis of derivatised steroids by matrix-assisted laser desorption/ionisation and post-source decay mass spectrometry

Neutral steroids are difficult to analyse using desorption ionisation methods coupled with mass spectrometry (MS). However, steroids with an unhindered ketone group can readily be derivatised with the Girard P (GP) reagent to give GP hydrazones. Steroid GP hydrazones contain a quaternary nitrogen atom and are readily desorbed in the matrix-assisted laser desorption/ionisation (MALDI) process, giving an improvement in sensitivity of two orders of magnitude. Steroids without a ketone group, but with a 3beta-hydroxy-Delta5 function, can be readily converted to 3-oxo-Delta4 steroids and subsequently derivatised to GP hydrazones for MALDI analysis. In addition to giving strong [M]+ ions upon MALDI, steroid GP hydrazones give informative post-source decay (PSD) spectra. By using the accurate mass of the precursor-ion measured by MALDI-MS, in combination with the structural information encoded in its PSD spectrum, steroid structures can readily be determined.     

Specific peptide patterns of follicular fluids at different growth stages analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Human follicular fluid (HFF) has been suggested to influence oocyte development potential, and some of HFF proteins may be potential markers for oocyte maturation during follicular development. Using matrix-assisted laser desorption /ionization-time of flight mass spectrometry (MALDI-TOF MS), the presence of specific peptide peaks in HFF which could represent the follicle development potential was evaluated. HFF from different developmental stages were first digested and the resultant peptide mixtures were directly analyzed by MALDI-TOF MS. It was shown that the frequencies of specific peaks demonstrated higher reproducibility than peak intensities after multiple measurements (>or=6 times) per sample. Using this approach, a reliable peak list for each different sample could be generated by combining the information from multiple measurements. By comparing the peak lists from different samples at different growth stages, we found that 5 specific peaks appeared in the 100% frequency category of 6 replicates in all the HFF samples containing mature oocyte. Similarly, such 25 peptide peaks were also identified for HFF containing immature oocyte. These specific peaks could be used to distinguish HFF from different stages as biomarkers related to follicle development and maturation. After searching the protein database, some proteins that are known to be involved in the development and maturation of oocyte were identified, such as apolipoprotein A-I, collagen type IV, integrin, et al. Identification of such proteins in our experiment further proved that the direct analysis of tryptic digests could be of practical value.     

Label-free measurement of histone lysine methyltransferases activity by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Histone lysine methyltransferases (HKMTs) are enzymes that play an essential role in epigenetic regulation. Thus, identification of inhibitors specifically targeting these enzymes represents a challenge for the development of new antitumor therapeutics. Several methods for measuring HKMT activity are already available. Most of them use indirect measurement of the enzymatic reaction through radioactive labeling or antibody-recognized products or coupled enzymatic assays. Mass spectrometry (MS) represents an interesting alternative approach because it allows direct detection and quantification of enzymatic reactions and can be used to determine kinetics and to screen small molecules as potential inhibitors. Application of mass spectrometry to the study of HKMTs has not been fully explored yet. We describe here the development of a simple reliable label-free MALDI-TOF MS-based assay for the detection and quantification of peptide methylation, using SET7/9 as a model enzyme. Importantly, the use of expensive internal standard often required in mass spectrometry quantitative analysis is not necessary in this assay. This MS assay allowed us to determine enzyme kinetic parameters as well as IC₅₀ for a known inhibitor of this enzyme. Furthermore, a comparative study with an antibody-based immunosorbent assay showed that the MS assay is more reliable and suitable for the screening of inhibitors.     

Bacteriocin detection from whole bacteria by matrix-assisted laser desorption ionization-time of flight mass spectrometry

Class I bacteriocins (lantibiotics) and class II bacteriocins are antimicrobial peptides secreted by gram-positive bacteria. Using two lantibiotics, lacticin 481 and nisin, and the class II bacteriocin coagulin, we showed that bacteriocins can be detected without any purification from whole producer bacteria grown on plates by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). When we compared the results of MALDI-TOF-MS performed with samples of whole cells and with samples of crude supernatants of liquid cultures, the former samples led to more efficient bacteriocin detection and required less handling. Nisin and lacticin 481 were both detected from a mixture of their producer strains, but such a mixture can yield additional signals. We used this method to determine the masses of two lacticin 481 variants, which confirmed at the peptide level the effect of mutations in the corresponding structural gene.     

Liquid ultraviolet matrix-assisted laser desorption/ionization -- mass spectrometry for automated proteomic analysis

We have combined several key sample preparation steps for the use of a liquid matrix system to provide high analytical sensitivity in automated ultraviolet -- matrix-assisted laser desorption/ionisation -- mass spectrometry (UV-MALDI-MS). This new sample preparation protocol employs a matrix-mixture which is based on the glycerol matrix-mixture described by Sze et al. The low-femtomole sensitivity that is achievable with this new preparation protocol enables proteomic analysis of protein digests comparable to solid-state matrix systems. For automated data acquisition and analysis, the MALDI performance of this liquid matrix surpasses the conventional solid-state MALDI matrices. Besides the inherent general advantages of liquid samples for automated sample preparation and data acquisition the use of the presented liquid matrix significantly reduces the extent of unspecific ion signals in peptide mass fingerprints compared to typically used solid matrices, such as 2,5-dihydroxybenzoic acid (DHB) or alpha-cyano-hydroxycinnamic acid (CHCA). In particular, matrix and low-mass ion signals and ion signals resulting from cation adduct formation are dramatically reduced. Consequently, the confidence level of protein identification by peptide mass mapping of in-solution and in-gel digests is generally higher.     

A quantitative method for the analysis of glycated and glutathionylated hemoglobin by matrix-assisted laser desorption ionization-time of flight mass spectrometry

The quantization of glycated isoforms of hemoglobin has been increasingly used in clinical practice in recent years. Glycated hemoglobin is currently considered the most important measurement for long-term control of the glycemic state and it has become a reference tool for the management of diabetes. Glutathionylated hemoglobin is an increasingly clinically relevant covalent adduct of glutathione with beta chain of the globin and its concentration has been correlated with oxidative stress. We have developed an innovative technique based on linear mode matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry for quantitative analysis of hemoglobin species. This method was applied to the quantification of glycated and glutathionylated hemoglobin. A rigorous comparison was pursued to evaluate the analytical performances in quantifying glycated hemoglobin in comparison to an established high-performance liquid chromatography method. Our results indicated a complete equivalence between the two methods. The same analysis enabled the quantitative determination of the glutathionylated hemoglobin fraction. This isoform was investigated in an adult Italian population (184 individuals, 101 males and 83 females), indicating a bimodal distribution of this species. In fact 65.22% of screened individuals had glutathionylated hemoglobin levels lower than 0.50% while 34.78% had glutathionylated hemoglobin levels higher than 0.50%. A semiautomatic robotic procedure was developed for fast analysis of a large number of samples. This is the first report of a quantitative application of linear MALDI-TOF mass spectrometry for the determination of glutathionylated hemoglobin in blood samples. This method allows fast screening of this hemoglobin isoform, therefore opening the route to explore its specificity and sensitivity as a molecular biomarker.     

High-throughput phospholipid quantitation in mammalian cells using matrix-assisted laser desorption ionization-time of flight mass spectrometry with N-trifluoroacetyl-phosphatidylethanolamine as internal standard

A high-throughput method is described for quantitative analysis of phospholipids. The method comprises extraction of lipids, addition of the internal standard N-trifluoroacetyl-phosphatidylethanolamine, and final analysis using matrix-assisted laser desorption ionization mass spectrometry. Quantitative data are obtained by calibration directly in the sample matrix. Calibration with one phosphatidylcholine was found sufficient for quantification of all major phosphatidylcholines tested. The method is very sensitive, has broad application, and is easily applicable to any biological sample. The detection limit for phosphatidylcholines was clearly below 2 μg on the spot, requiring less than 4000 cells corresponding to about 1.6 μg cell dry mass.