Quality control based on isotopic distributions for high-throughput MALDI-TOF and MALDI-FTICR serum peptide profiling

In this study, we have implemented a new quality control (QC) parameter for peptide profiling based on isotopic distributions. This QC parameter is an objective measure and facilitates automatic sorting of large numbers of peptide spectra. Peptides in human serum samples were enriched using reversed-phase C₁₈-functionalized magnetic beads using a high-throughput robotic platform. High-resolution MALDI-TOF and ultrahigh resolution MALDI-FTICR mass spectra were obtained and a workflow was developed for automated analysis and evaluation of these profiles. To this end, the isotopic distributions of multiple peptides were quantified from both MALDI-TOF and MALDI-FTICR spectra. Odd peptide isotope distributions in TOF spectra could be rationalized from ultrahigh resolution FTICR spectra that showed overlap of different peptides. The comparison of isotope patterns with estimated polyaveragine distributions was used to calculate a QC value for each single mass spectrum. Sorting these QC values enabled the best MALDI spectrum to be selected from replicate spots. Moreover, using this approach spectra containing high intensities of polymers or other contaminants and lacking peptides of interest can be efficiently removed from a clinical dataset. In general, this method simplifies the exclusion of low quality spectra from further statistical analysis.     

An improved calibration method for the matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resononance analysis of 15N-metabolically- labeled proteome digests using a mass difference approach

High mass measurement accuracy of peptides in enzymatic digests is critical for confident protein identification and characterization in proteomics research. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) can provide low or sub-ppm mass accuracy and ultrahigh resolving power. While for ESI-FT-ICR-MS, the mass accuracy is generally 1 ppm or better, with matrix-assisted laser desorption/ionization (MALDI)-FT-ICR-MS, the mass errors can vary from sub-ppm with internal calibration to over 100 ppm with conventional external calibration. A novel calibration method for (15)N-metabolically labeled peptides from a batch digest of a proteome is described which corrects for space charge induced frequency shifts in FT-ICR spectra without using an internal calibrant. This strategy utilizes the information from the mass difference between the (14)N/(15)N peptide peak pairs to correct for space charge induced mass shifts after data collection. A procedure for performing the mass correction has been written into a computer program and has been successfully applied to high-performance liquid chromatography-MALDI-FT- ICR-MS measurement of (15)N-metabolic labeled proteomes. We have achieved an average measured mass error of 1.0 ppm and a standard deviation of 3.5 ppm for 900 peptides from 68 MALDI-FT-ICR mass spectra of the proteolytic digest of a proteome from Methanococcus maripaludis.     

基质中加入少量盐类改善MALDI-TOF MS的谱图信噪比

自20世纪80年代发明基质辅助激光解吸电离(Matrix assisted laser desorption ionization,MALDI)质谱以来,该技术已在生物分子分析方面得到了广泛应用．作为一种离子化方法,MALDI具有灵敏度高,对样品要求低,能耐高浓度盐和缓冲剂等优点．测定过程中使用合适的基质不仅能提高测试灵敏度和分辨率,还能扩增测试样品的种类。     

Production of Reliable MALDI Spectra with Quality Threshold Clustering of Replicates

We present the first application of the quality threshold (QT) clustering algorithm to mass spectrometry (MS) data. The unique abilities of QT clustering to yield precision nodes that are commensurate with the mass measurement precision of the instrument are exploited to generate a consensus spectrum out of multiple replicate spectra. The spectral dot product and confidence intervals are used as a tool for evaluating the similarity and reproducibility between the consensus and replicates. The method is equally applicable to high and low resolution measurements. This paper demonstrates applications to linear spectra from a matrix assisted laser desorption ionization (MALDI) time of flight (TOF) instrument as well as peptide fragmentation data obtained from a TOF/TOF after unimolecular decomposition. The advantages of clustering to mitigate the inherent precision the shortcomings of MALDI data are discussed.     

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.     

BSE control: detection of gelatine-derived peptides in animal feed by mass spectrometry

The epidemic of bovine spongiform encephalopathy (BSE) is thought to have resulted from feeding scrapie-infected sheep to cattle. This has led to a ban of feeding animals with "processed animal protein"(PAP). We report a novel approach for the mass spectrometric detection of PAP contamination in animal feedstuffs by detecting gelatine, a derivative of the major animal protein collagen. A method was developed to hydrolyse gelatine standards with hydrochloric acid, followed by detection of the derived hydrolytic peptides at m/z 828, 915, 957 and 1044 by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and liquid chromatography electrospray ionisation mass spectrometry (LC-ESI-MS-MS). The marker peptides can be detected at concentrations of 100 ng ml(-1) gelatine in water with MALDI. The procedure was adapted to obtain a suitable peptide map of gelatine extracted from spiked feed. The ratio of signal area of the gelatine-derived peptide at m/z 1044 to the internal standard at m/z 556 is shown to relate to the total amount of gelatine present in the sample.     

基质辅助激光解吸电离飞行时间质谱研究2: 四硫 富瓦烯化合物

采用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS),对四硫富瓦烯化合物进行质谱表征。在所用的实验条件下,样品很容易解吸电离生成单电荷分子离子,得到单同位素分辨的质谱图。26种实际样品的质谱分析结果表明;MALDI-TOF-MS可以比其它质谱方法更有效、更方便地用于此类化合物的质谱分析,解决了此类化合物不易进行质谱鉴定的难题。     

MALDI‐TOF‐Massenspektrometrie: Risikoanalyse im Flug

The high accuracy, molecular resolution and sensitivity of matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) make it an efficient method for analysing all kinds of biomolecules including nucleic acids, proteins/peptides, carbohydrates and lipids. MALDI‐TOF‐MS based high‐throughput genotyping of genetic heterogeneities possesses the potential of becoming a routine method. MAL‐DI‐TOF‐MS can be used for the identification of proteins and posttranslational modifications. Taken together, MALDI‐TOF‐MS represents a integrated platform technology in bioanalytics and molecular medicine.     

Matrix-free mass spectrometric imaging using laser desorption ionisation Fourier transform ion cyclotron resonance mass spectrometry

Mass spectrometry imaging (MSI) is a powerful tool in metabolomics and proteomics for the spatial localization and identification of pharmaceuticals, metabolites, lipids, peptides and proteins in biological tissues. However, sample preparation remains a crucial variable in obtaining the most accurate distributions. Common washing steps used to remove salts, and solvent-based matrix application, allow analyte spreading to occur. Solvent-free matrix applications can reduce this risk, but increase the possibility of ionisation bias due to matrix adhesion to tissue sections. We report here the use of matrix-free MSI using laser desorption ionisation performed on a 12 T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. We used unprocessed tissue with no post-processing following thaw-mounting on matrix-assisted laser desorption ionisation (MALDI) indium-tin oxide (ITO) target plates. The identification and distribution of a range of phospholipids in mouse brain and kidney sections are presented and compared with previously published MALDI time-of-flight (TOF) MSI distributions.     

Identification of mammalian cell lines using MALDI-TOF and LC-ESI-MS/MS mass spectrometry

Direct mass spectrometric analysis of complex biological samples is becoming an increasingly useful technique in the field of proteomics. Matrix-assisted laser desorption/ionization mass spectroscopy (MALDI-MS) is a rapid and sensitive analytical tool well suited for obtaining molecular weights of peptides and proteins from complex samples. Here, a fast and simple approach to cellular protein profiling is described in which mammalian cells are lysed directly in the MALDI matrix 2,5-dihydroxybenzoic acid (DHB) and mass analyzed using MALDI-time of flight (TOF). Using the unique MALDI mass spectral "fingerprint" generated in these analyses, it is possible to differentiate among several different mammalian cell lines. A number of techniques, including MALDI-post source decay (PSD), MALDI tandem time-of-flight (TOF-TOF), MALDI-Fourier transform ion cyclotron resonance (FTICR), and nanoflow liquid chromatography followed by electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS) were employed to attempt to identify the proteins represented in the MALDI spectra. Performing a tryptic digestion of the supernatant of the cells lysed in DHB with subsequent LC-ESI-MS/MS analysis was by far the most successful method to identify proteins.     

Targeted tandem mass spectrometry for high-throughput comparative proteomics employing NanoLC-FTICR MS with external ion dissociation

Targeted tandem mass spectrometry (MS/MS) is an attractive proteomic approach that allows selective identification of peptides exhibiting abundance differences, e.g., between culture conditions and/or diseased states. Herein, we report on a targeted LC-MS/MS capability realized with a hybrid quadrupole-7 tesla Fourier transform ion cyclotron resonance (FTICR) mass spectrometer that provides data-dependent ion selection, accumulation, and dissociation external to the ICR trap, and a control software that directs intelligent MS/MS target selection based on LC elution time and m/z ratio. We show that the continuous on-the-fly alignment of the LC elution time during the targeted LC-MS/MS experiment, combined with the high mass resolution of FTICR MS, is crucial for accurate selection of targets, whereas high mass measurement accuracy MS/MS data facilitate unambiguous peptide identifications. Identification of a subset of differentially abundant proteins from Shewanella oneidensis grown under suboxic versus aerobic conditions demonstrates the feasibility of such approach.     

Enhancement of matrix-assisted laser desorption/ionization photodissociation tandem time-of-flight mass spectra; spectral reduction and cleanup of isotopomeric contamination

Weak signal intensity and poor precursor ion selection are the major difficulties in tandem time-of-flight (TOF) mass spectrometry of ions generated by matrix-assisted laser desorption/ionization (MALDI). Even though the latter can be overcome in photodissociation (PD) tandem TOF mass spectrometry via ion pulse-PD laser pulse synchronization, clean monoisotopic selection of precursor ions of high m/z can often be difficult for various reasons. A considerable enhancement of post-source decay (PSD) and PD tandem mass spectra has been achieved in this work via single-ion detection and post-acquisition reduction of the spectra. Also, an algorithm has been developed to clean up isotopomeric contamination when the resolution for precursor ion selection is less than adequate. A high-quality tandem TOF mass spectrum which results from PD of virtually monoisotopic precursor ions has been obtained.     

Characterization and de novo sequencing of Atlantic salmon vitellogenin protein by electrospray tandem and matrix-assisted laser desorption/ionization mass spectrometry

Vitellogenin (VTG) is a protein produced by the liver of oviparous animals in response to circulating estrogens. In the plasma of males and immature females, VTG is undetectable. VTG has been used as a biomarker for exposure to endocrine disruptors in many species. In the present study, characterization of intact Atlantic salmon VTG was effected using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI ToF MS). Tryptic digest peptides were analyzed by MALDI ToF MS to obtain a peptide mass fingerprint. De novo sequencing of the tryptic peptides used low-energy collisionally-induced dissociation (CID) in an electrospray ionization quadrupole-ToF orthogonal hybrid mass spectrometer (ESI Q-ToF MS/MS). The interpretation of the product-ion spectra obtained from the ESI Q-ToF MS/MS was done by Lutefisk, a computer-based software algorithm. The molecular mass of the intact protein was found to be 187335 Da. A total of 14 tryptic peptides were sequenced and compared with the complete rainbow trout VTG and the partial Atlantic salmon VTG sequences found in the Swiss-Prot database. De novo sequencing by CID MS/MS of 11 Atlantic salmon tryptic digest peptides with selected precursor ions at m/z 788.24, 700.20, 794.75, 834.31, 889.28, 819.79, 865.27, 843.81, 572.20, 573.66 and 561.68 showed high homology with the known sequence of rainbow trout VTG. The last two precursor peptide ions, found at m/z 573.66 and m/z 561.68, also specifically matched the known portion of the Atlantic salmon VTG sequence. Finally, three tryptic precursor peptide ions found at m/z 795.18, 893.28 and 791.05, provided product-ion spectra, which were exclusive to the unsequenced portion of the Atlantic salmon VTG.     

Quantitative mapping of glycoprotein micro‐heterogeneity and macro‐heterogeneity: an evaluation of mass spectrometry signal strengths using synthetic peptides and glycopeptides

Mass spectrometry (MS) is used to quantify the relative distribution of glycans attached to particular protein glycosylation sites (micro‐heterogeneity) and evaluate the molar site occupancy (macro‐heterogeneity) in glycoproteomics. However, the accuracy of MS for such quantitative measurements remains to be clarified. As a key step towards this goal, a panel of related tryptic peptides with and without complex, biantennary, disialylated N‐glycans was chemically synthesised by solid‐phase peptide synthesis. Peptides mimicking those resulting from enzymatic deglycosylation using PNGase F/A and endo D/F/H were synthetically produced, carrying aspartic acid and N‐acetylglucosamine‐linked asparagine residues, respectively, at the glycosylation site. The MS ionisation/detection strengths of these pure, well‐defined and quantified compounds were investigated using various MS ionisation techniques and mass analysers (ESI‐IT, ESI‐Q‐TOF, MALDI‐TOF, ESI/MALDI‐FT‐ICR‐MS). Depending on the ion source/mass analyser, glycopeptides carrying complex‐type N‐glycans exhibited clearly lower signal strengths (10–50% of an unglycosylated peptide) when equimolar amounts were analysed. Less ionisation/detection bias was observed when the glycopeptides were analysed by nano‐ESI and medium‐pressure MALDI. The position of the glycosylation site within the tryptic peptides also influenced the signal response, in particular if detected as singly or doubly charged signals. This is the first study to systematically and quantitatively address and determine MS glycopeptide ionisation/detection strengths to evaluate glycoprotein micro‐heterogeneity and macro‐heterogeneity by label‐free approaches. These data form a much needed knowledge base for accurate quantitative glycoproteomics. Copyright © 2013 John Wiley & Sons, Ltd.     

Urinary peptides as a diagnostic tool for renal failure detected by matrix-assisted laser desorption/ionisation mass spectrometry: an evaluation of their clinical significance

The development of new analytical methodologies related to the proteome for the evaluation of renal physiology and pathology is surely of wide interest for physicians, giving them new tools for monitoring complications associated with diabetes, such as end-stage renal disease. In the present study, the clinical significance of the urinary abundance of two peptides, SGSVIDQSRVLNLGPITR (the uromodulin precursor, m/z 1912) and IGPHypGPHypGLMGPP [present in the collagen-α-5(IV) chain precursor, m/z 1219], detected by matrix- assisted laser desorption/ionisation mass spectrometry (MALDI/MS) in microalbuminuric or nephropathic diabetic patients and in non-diabetic nephropathic patients was evaluated. A progressive increase in the abundance of the ion at m/z 1219 and a decrease in the abundance of the ion at m/z 1912 have been found in diabetic microalbuminuric, diabetic-nephropathic and nephropathic patients. Linear correlations are present between serum creatinine values and the abundances of the ions at m/z 1219 (positive correlation, r=0.3645, P<0.0001) and at m/z 1912 (negative correlation, r=-0.3053, P<0.0005). Correlations between the MALDI data and the estimated glomerular filtration rate were also found, while relationships with urinary albumin excretion were found only in sub-sets of patients. Analysis of receiver operating characteristic curves showed a sensitivity up to 96% and a specificity of up to 84% for the two ionic species, or their ratio, for distinguishing diabetic patients with different degrees of nephropathy from healthy subjects, proving that the urinary abundance of the two peptides at m/z 1219 and m/z 1912, determined with MALDI/MS, may be considered as a possible diagnostic tool for the determination of progression toward renal failure, also with the aim of monitoring kidney function, in diabetic patients.     

Serum protein profiling in patients with inflammatory bowel diseases using selective solid-phase bulk extraction, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and chemometric data analysis

The identification of new biomarkers or a disease-related protein fingerprint for inflammatory bowel diseases (IBDs) represents a major task in the diagnosis, prognosis and pharmacological therapy. To address these issues, a simple and rapid analytical proteomic method for serum protein profiling based on selective beads-based solid-phase bulk extraction, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and chemometric data analysis was developed. Serum proteins from healthy subjects (22) and patients with Crohn's disease (15) and ulcerative colitis (26) were selectively extracted according to reversed-phase (C18), strong anion-exchange (SAX) and metal ion affinity (IDA-Cu(II)) principles. This approach allowed enrichment of serum proteins/peptides due to the high interaction surface between analytes and the solid phase and high recovery due to the elution step performed directly on the MALDI-target plate. The MALDI-TOF MS serum protein profiles were acquired and, after a data pre-processing step, analyzed by linear discriminant analysis (LDA), a chemometric classification technique, in order to classify serum samples among healthy subjects and patients with inflammatory bowel diseases (IBDs). Since the high number of variables in the MALDI spectra (more than 16000 m/z values) prevents the use of LDA, the variables were reduced to 10-20 by features selection, thus allowing the evaluation of a pattern of m/z values with high discriminant power. Serum protein profiles obtained by reversed-phase extraction and the selection of 20 m/z values gave the best overall prediction ability (96.9%). The recognition of these m/z values may allow the identification of protein biomarkers involved in IBDs.     

Mass correlated acceleration in a reflectron MALDI TOF mass spectrometer: an approach for enhanced resolution over a broad mass range

Compared to continuous extraction, pulsed extraction (PE) of ions formed by matrix-assisted laser desorption/ionization (MALDI) in time-of-flight (TOF) mass spectrometers significantly improves mass resolution. Parameters such as extraction voltage, delay time, and correction pulse must be varied, however, to achieve optimum mass resolution over a broad mass range because the PE method is mass dependent. We previously reported a novel method, mass correlated acceleration (MCA), which we have now combined with a reflectron MALDI TOF mass spectrometer to further enhance mass resolution over a broader mass range. Unlike the PE method, MCA is not mass dependent and high resolution mass spectra can be achieved with a single tuning of instrument parameters. The ions may be brought into focus simultaneously, i.e., the multi-channel recording advantage can be more fully realized. The MCA dual-stage ion source design includes an extraction pulse region and an acceleration region that contains a time-dependent waveform correlated with mass. We demonstrate the validity of this novel technique with applications in peptide mixture analysis and protein digests of lysozyme and bovine serum albumin.     

Automatic internal calibration in liquid chromatography/Fourier transform ion cyclotron resonance mass spectrometry of protein digests

Accurately measured peptide masses can be used for large-scale protein identification from bacterial whole-cell digests as an alternative to tandem mass spectrometry (MS/MS) provided mass measurement errors of a few parts-per-million (ppm) are obtained. Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) routinely achieves such mass accuracy either with internal calibration or by regulating the charge in the analyzer cell. We have developed a novel and automated method for internal calibration of liquid chromatography (LC)/FTICR data from whole-cell digests using peptides in the sample identified by concurrent MS/MS together with ambient polydimethylcyclosiloxanes as internal calibrants in the mass spectra. The method reduced mass measurement error from 4.3 +/- 3.7 ppm to 0.3 +/- 2.3 ppm in an E. coli LC/FTICR dataset of 1000 MS and MS/MS spectra and is applicable to all analyses of complex protein digests by FTICRMS.     

An investigation on the nature of the peptide at m/z 904, overexpressed in plasma of patients with colorectal cancer and familial adenomatous polyposis

In an investigation devoted to the search for plasma markers for colorectal cancer (CRC), carried out by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, a series of overexpressed peptides were identified in the plasma of patients. Among them the peptide with molecular weight 903 Da was the most abundant one, with a mean +/- (SD) relative abundance of 37 +/- 17% and a frequency over 60%. Interestingly, also in plasma samples of ten subjects affected by familial adenomatous polyposis (FAP), the peptide with molecular weight 903 was overexpressed. In this investigation, MALDI/MS/MS experiments were carried out on the ion at m/z 904 detected in the MALDI mass spectra of CRC and FAP patients. The data analysis by SwissProt.2007.01.09 indicates that this peptide is due to the sequence RPPGFSPF, found in the kininogen-1 precursor, which is an alpha-2-thiol proteinase inhibitor. In the case of subjects affected by a particular FAP syndrome, the MALDI/MS/MS spectra were quite different from those obtained from CRC and FAP patients. In fact, two sequences have been evidenced: RPPGFSPF belonging to kininogen-1 precursor, and PRKSSSSR belonging to Forkhead box protein 01A.     

Fast analysis of low molecular mass compounds present in snake venom: identification of ten new pyroglutamate-containing peptides

Characterization of the peptide content in snake venoms can be an important tool for the investigation of new pharmacological lead compounds. For this purpose, single-step analysis of crude venoms has recently been demonstrated using mass spectrometry (MS) techniques. Reproducible profiles of ions in MS and MS/MS spectra may also be used to compare venoms from different species. In this work matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was used to obtain mass patterns of the major peptides (<8 kDa) found in pooled venoms from the genera Bothrops and Crotalus. Venoms from five different Bothrops species (B. jararaca, B. insularis, B. alternatus, B. jararacussu, and B. neuwiedi) and three Crotalus species (C. viridis, C. adamanteus and C. durissus terrificus) were analyzed. In agreement with other reports, venoms from Bothrops species contained a variety of peptides in the range m/z 1000-1500, and in some samples larger components (m/z 7000-8000) were detected. In the Crotalus species venoms were rich in peptides ranging from m/z 1000-1500 and 4000-5500. MS/MS experiments on the low molecular mass peptides (m/z 1000-1500) confirmed the presence of ten new bradykinin-potentiating peptides among venoms from genera Bothrops and Crotalus. In order to determine whether additional peptides could be identified after partial purification, B. jararaca venom was subjected to size-exclusion chromatography on Sephacryl S-200, and two distinct low molecular mass pools were analyzed further by MALDI-TOFMS. No additional peptides were detected from the pool with masses below 2000 Da but a substantial improvement with better resolution was observed for the pool with masses above 7000 Da, indicating that complex samples such as crude snake venoms can be analyzed for low molecular mass peptides using a single-step procedure.