Matrix-assisted Laser Desorption/ionization Fourier Transform Mass Spectrometry Research Articles

Method development for compositional analysis of low molecular weight poly(vinyl acetate) by matrix-assisted/laser desorption-mass spectrometry and its application to analysis of chewing gum

The influence of the sample preparation parameters (the choice of the solvent and of the matrix:analyte ratio) was investigated and optimal conditions were established for MALDI mass spectrometry analysis of the pristine low molecular weight polyvinyl acetate (PVAc). It was demonstrated that comparison of polymer’s and solvent’s Hansen solubility parameters could be used as a guide when choosing the solvent for MALDI sample preparation. The highest intensity PVAc signals were obtained when ethyl acetate was used as a solvent along with the lowest matrix–analyte ratio (2,5-dihydroxybenzoic acid was used as a matrix in all experiments). The structure of the PVAc was established with high accuracy using the matrix-assisted laser desorption/ionization-Fourier transform mass spectrometry (MALDI-FTMS) analysis. It was demonstrated that PVAc undergoes unimolecular decomposition by losing acetic acid molecules from its backbone under the conditions of FTMS measurements. Number and weight average molecular weights as well as polydispersity indices were determined with both MALDI-TOF and MALDI-FTMS methods. The sample preparation protocol developed was applied to the analysis of a chewing gum and the molecular weight and structure of the polyvinyl acetate present in the sample were established. Thus, it was shown that optimized MALDI mass spectrometry could be used successfully for characterization of polyvinyl acetate in commercially available chewing gum.

Determination of cocaine, cocaine metabolites and cannabinoids in single hairs by MALDI Fourier transform mass spectrometry – preliminary results

In a preliminary test, four single hairs of a drug abuser were analyzed for the presence of drugs by MALDI Fourier transform mass spectrometry (MALDI-FTMS). Washed hair strains were directly fixed on a sample plate using pressure stable, double-sided adhesive tape; α-cyano-4-hydroxycinnamic acid matrix was manually spotted onto the hair strains. FTMS full scans were obtained moving from the hair root region towards the hair tip. Cocaine (accurate m/z ratio 304.15433) was identified mostly from the root of the hair and then later again towards the hair tip. This was confirmed by analysis of a second hair. Additionally cocaine metabolites with m/z ratio 290.13868 (benzoylecgonine), and m/z 318.16998 (cocaethylene) were detected for plausibility control. Using the MALDI technique, time-related information was obtained concerning the behavioural pattern of the consumer with high resolution compared to conventional procedures. However, in two hairs of the same individual which were analyzed under the same conditions, negative results were achieved. These preliminary results confirm the applicability of MALDI-MS for the determination of drugs and pharmaceuticals in hair samples being useful in forensic toxicology. The high chronological resolution allows an enhanced interpretation concerning the periods of drug administration. However, the negative results with two negative hairs have also demonstrated that hair analysis of single hairs can lead to misinterpretation. Different growth rates have to be considered, and particularly the phenomenon of different growth phases (anagen, catagen, telogen) require attention.

Study of Effects of Cigarette Smoke Condensates on Acetylcholinesterase Activity in Human Lung Epithelial Cells by Matrix-Assisted Laser Desorption/Ionization-Fourier Transform Mass Spectrometry

Matrix-Assisted Laser Desorption/Ionization-Fourier Transform Mass Spectrometry (MALDI-FTMS) is an emerging method for kinetic measurements and inhibitor screening of acetylcholinesterase (AChE). This method is accurate, reproducible, and was successfully applied to the analysis of various biosamples. Recent studies showed the important biological functions of acetylcholinesterase in many kinds of tumors caused by smoking; however, the effect of cigarette smoke condensates (CSCs) on AChE was unclear. Herein, the effects of CSCs on AChE activity in human lung epithelial cells were studied by MALDI-FTMS. The results showed that AChE activity in human lung epithelial cells could be inhibited by CSCs, and inhibition degree on AChE activity depended on the amount of CSCs. Owing to the close relationship of AChE to lung cancer caused by smoking, such findings provide information regarding the relation between the aggressiveness of lung tumor and cigarette smoking.

Rapid Detection of Irreversible Acetylcholineasterase Inhibitor by Mass Spectrometry Assay

AbstractHere we developed a rapid method to detect acetylcholinesterase (AChE) activity by matrix‐assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI‐FTMS) for screening irreversible AChE inhibitors. Due to its good salt‐tolerance and low sample consumption, MALDI‐FTMS could facilitate rapid detection, especially detection in real application. AChE activity was determined through calculating abundance of substrate and product in mass spectrometry. By this approach, we investigated the relation of organophosphorous (OP) concentrations and AChE inhibition. Shown in different inhibition curves from different OP pesticides, enzyme inhibitions still kept good correlation with concentration of OPs. Finally, this AChE‐inhibited method was applied to screen whole bloods of four decedents and discuss their death reason. In contrast to healthy persons, three of decedents showed low AChE activity, and probably died for irreversible AChE inhibitors. Through the following detecting in GC‐MS/MS, the possible death reason of these three decedents was confirmed, and another decedent actually died for sumicidin, a non‐AChE inhibitor. It demonstrated that screening irreversible AChE inhibitors by detecting enzyme activity in MALDI‐FTMS provided fast and accurate analysis results and excluded another toxicants not functioning on AChE. This method offered alternative choices for indicating the existence of enzyme inhibitors.

Urinary Metabolomics in Monitoring Acute Tubular Injury of Renal Allografts: A Preliminary Report

Acute tubular injury (ATI) is very common in biopsy specimens from renal allografts that suffer from delayed graft function (DGF) or dysfunction. Currently there are few reports on investigating small molecule metabolites in urine samples from transplant recipients as a noninvasive method to predict the ATI of renal allografts instead of an allograft biopsy. In our study matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) was used to analyze small molecule metabolites in urine samples from renal transplant recipients with biopsy-proven slight ATI or moderate ATI or acute tubular necrosis (ATN). To evaluate the ATI-specific value of those small molecules, we applied the Principal Component Analysis (PCA) program. Mass spectra data were imported into the PCA, where loading graphs were constructed to express the constituents of the urine samples. Slight ATI, moderate ATI, or ATN of renal allografts were separated obviously in the loading graph. The position of urine samples in the graph may reflect the tubular injury status of allografts. A farther apart point from the original site may mean the allograft suffered from more severe ATI (even ATN), and vice versa. Detection of small molecule metabolites in urine samples of recipients through MALDI-FTMS may offer a promising noninvasive, high throughput, rapid tool to predict ATI/ATN of renal allografts.

Assisted inhibition effect of acetylcholinesterase with n-octylphosphonic acid and application in high sensitive detection of organophosphorous pesticides by matrix-assisted laser desorption/ionization Fourier transform mass spectrometry

A simple and practical approach to improve the sensitivity of acetylcholinesterase (AChE)-inhibited method has been developed for monitoring organophosphorous (OP) pesticide residues. In this work, matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) was used to detect AChE activity. Due to its good salt-tolerance and low sample consumption, MALDI-FTMS facilitates rapid and high-throughput screening of OP pesticides. Here we describe a new method to obtain low detection limits via employing external reagents. Among candidate compounds, n-octylphosphonic acid ( n-Octyl-PA) displays assistant effect to enhance AChE inhibition by OP pesticides. In presence of n-Octyl-PA, the percentages of AChE inhibition still kept correlation with OP pesticide concentrations. The detection limits were improved significantly even by 10 2–10 3 folds in comparison with conventional enzyme-inhibited methods. Different detection limits of OP pesticides with different toxicities were as low as 0.005 μg L −1 for high toxic pesticides and 0.05 μg L −1 for low toxic pesticides. Besides, the reliability of results from this method to analyze cowpea samples had been demonstrated by liquid-chromatography tandem mass spectrometry (LC–MS/MS). The application of this commercial available assistant agent shows great promise to detect OP compounds in complicated biological matrix and broadens the mind for high sensitivity detection of OP pesticide residues in agricultural products.

Determination of β-Lactamase Residues in Milk Using Matrix-Assisted Laser Desorption/Ionization Fourier Transform Mass Spectrometry

A selective, fast, and effective enzyme assay based on matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) for quantifying beta-lactamase, an illegal additive in milk products, has been reported. The strengths of the mass spectrometric assay are its response to all substrate and products, simple and direct detection of the conversion of substrate, and facile determination of enzyme activity. Also, MALDI MS is tolerant to many buffer salts and reagents without the requirement of complicated sample pretreatment procedures. In this study, the approach was used to detect the presence of beta-lactamases (BLA) in milk samples. The amount of BLA that could be determined in a milk sample is 6 x 10(-3) U x mL(-1) by this approach. To test the strategy, it has been applied to the fortified milk (adding a BLA product known as an antimicrobial destroyer). It is then tested whether the pasteurization procedure of the milk process affects the activity of BLA in milk samples. This study offers a perspective into the utility of MALDI-FTMS as an alternative detection tool for BLA screening in milk.

Immunoaffinity-based mass spectrometric characterization of the FMRFamide-related peptide family in the pericardial organ of Cancer borealis

The tetrapeptide, FMRFamide, was first discovered in 1977 in the molluscan nervous system and was found to affect the contractile force of molluscan cardiac muscle and other muscles [1]. Since then, numerous FMRFamide-related peptides (FaRPs) have been reported in both invertebrate and vertebrate species [2–9]. We have previously reported the detection and identification of numerous FaRPs in Cancer borealis pericardial organs (POs), one of the major neurosecretory structures in the crustaceans [2,3]. Here, we have developed two immunoaffinity-based methods, immunoprecipitation (IP) and immuno-dot blot screening assay, for the enrichment of FaRPs in C. borealis POs. A combined mass spectrometry (MS)-based approach involving both matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) and nanoscale liquid chromatography coupled to electrospray ionization quadrupole time-of-flight tandem mass spectrometry (nanoLC-ESI-QTOF MS/MS) is used for a more comprehensive characterization of the FaRP family by utilizing high mass accuracy measurement and efficient peptide sequencing. Overall, 17 FMRFamide-related peptides were identified using these two complementary immuno-based approaches. Among them, three novel peptides were reported for the first time in this study.

Identification of SYWKQCAFNAVSCFamide: a broadly conserved crustacean C-type allatostatin-like peptide with both neuromodulatory and cardioactive properties

The allatostatins comprise three structurally distinct peptide families that regulate juvenile hormone production by the insect corpora allata. A-type family members contain the C-terminal motif -YXFGLamide and have been found in species from numerous arthropod taxa. Members of the B-type family exhibit a -WX(6)Wamide C-terminus and, like the A-type peptides, appear to be broadly conserved within the Arthropoda. By contrast, members of the C-type family, typified by the unblocked C-terminus -PISCF, a pyroglutamine blocked N-terminus, and a disulfide bridge between two internal Cys residues, have only been found in holometabolous insects, i.e. lepidopterans and dipterans. Here, using transcriptomics, we have identified SYWKQCAFNAVSCFamide (disulfide bridging predicted between the two Cys residues), a known honeybee and water flea C-type-like peptide, from the American lobster Homarus americanus (infraorder Astacidea). Using matrix assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS), a mass corresponding to that of SYWKQCAFNAVSCFamide was detected in the H. americanus brain, supporting the existence of this peptide and its theorized structure. Furthermore, SYWKQCAFNAVSCFamide was detected by MALDI-FTMS in neural tissues from five additional astacideans as well as 19 members of four other decapod infraorders (i.e. Achelata, Anomura, Brachyura and Thalassinidea), suggesting that it is a broadly conserved decapod peptide. In H. americanus, SYWKQCAFNAVSCFamide is capable of modulating the output of both the pyloric circuit of the stomatogastric nervous system and the heart. This is the first demonstration of bioactivity for this peptide in any species.

Expanding the Crustacean Neuropeptidome Using a Multifaceted Mass Spectrometric Approach

Jonah crab Cancer borealis is an excellent, long-served model organism for many areas of physiology, including the study of endocrinology and neurobiology. Characterizing the neuropeptides present in its nervous system provides the first critical step toward understanding the physiological roles of these complex molecules. Multiple mass spectral techniques were used to comprehensively characterize the neuropeptidome in C. borealis, including matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS), MALDI time-of-flight (TOF)/TOF MS and nanoflow liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (nanoLC-ESI-Q-TOF MS/MS). To enhance the detection signals and expand the dynamic range, direct tissue analysis, tissue extraction, capillary electrophoresis (CE) and off-line HPLC separation have also been employed. In total, 142 peptides were identified, including 85 previously known C. borealis peptides, 22 peptides characterized previously from other decapods, but new to this species, and 35 new peptides de novo sequenced for the first time in this study. Seventeen neuropeptide families were revealed including FMRFamide-related peptide (FaRP), allatostatin (A and B type), RYamide, orcokinin, orcomyotropin, proctolin, crustacean cardioactive peptide (CCAP), crustacean hyperglycemic hormone precursor-related peptide (CPRP), crustacean hyperglycemic hormone (CHH), corazonin, pigment-dispersing hormone (PDH), tachykinin, pyrokinin, SIFamide, red pigment concentrating hormone (RPCH) and HISGLYRamide. Collectively, our results greatly increase the number and expand the coverage of known C. borealis neuropeptides, and thus provide a stronger framework for future studies on the physiological roles played by these molecules in this important model organism.

Prion Disease Diagnosis by Proteomic Profiling

Definitive prion disease diagnosis is currently limited to postmortem assay for the presence of the disease-associated proteinase K-resistant prion protein. Using cerebrospinal fluid (CSF) from prion-infected hamsters, matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS), and support vector machines (SVM), we have identified peptide profiles characteristic of disease state. Using 10-fold leave-one-out cross-validation, we report a predictive accuracy of 72% with a true positive rate of 73% and a false positive rate of 27% demonstrating the suitability of using proteomic profiling and CSF for the development of multiple marker diagnostics of prion disease.

Lipid and Phospholipid Profiling of Biological Samples Using MALDI Fourier Transform Mass Spectrometry

Here we describe a study of the feasibility of lipid and phospholipid (PL) profiling using matrix assisted laser desorption/ionization (MALDI) Fourier transform mass spectrometry (FTMS) for two different applications. In this work PL profiles of different mammalian tissues as well as those of whole cell organisms were examined. In particular, comparative analysis of lipid and PL profiles of tissues from mice fed different diets was done and, in another application, MALDI FTMS was used to analyze PL profiles of genetically modified Saccharomyces cerevisiae. Computational sorting of the observed ions was done in order to group the lipid and PL ions from complex MALDI spectra. The PL profiles of liver tissues from mice fed different diets showed a cross correlation coefficient of 0.2580, indicating significant dissimilarity, and revealed more than 30 significantly different peaks at the 99.9% confidence level. Histogram plots derived from the spectra of wild type and genetically modified yeast resulted in a cross correlation coefficient 0.8941 showing greater similarity, but still revealing a number of significantly different peaks. Based on these results, it appears possible to use MALDI FTMS to identify PLs as potential biomarkers for metabolic processes in whole cells and tissues.

Monitoring Enzyme Reaction and Screening of Inhibitors of Acetylcholinesterase by Quantitative Matrix-Assisted Laser Desorption/Ionization Fourier Transform Mass Spectrometry

A matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS)-based assay was developed for kinetic measurements and inhibitor screening of acetylcholinesterase. Here, FTMS coupled to MALDI was applied to quantitative analysis of choline using the ratio of choline/acetylcholine without the use of additional internal standard, which simplified the experiment. The Michaelis constant (K(m)) of acetylcholinesterase (AChE) was determined to be 73.9 micromol L(-1) by this approach. For Huperzine A, the linear mixed inhibition of AChE reflected the presence of competitive and noncompetitive components. The half maximal inhibitory concentration (IC(50)) value of galantamine obtained for AChE was 2.39 micromol L(-1). Inhibitory potentials of Rhizoma Coptidis extracts were identified with the present method. In light of the results the referred extracts as a whole showed inhibitory action against AChE. The use of high-resolution FTMS largely eliminated the interference with the determination of ACh and Ch, produced by the low-mass compounds of chemical libraries for inhibitor screening. The excellent correlation with the reported kinetic parameters confirms that the MS-based assay is both accurate and precise for determining kinetic constants and for identifying enzyme inhibitors. The obvious advantages were demonstrated for quantitative analysis and also high-throughput characterization. This study offers a perspective into the utility of MALDI-FTMS as an alternate quantitative tool for inhibitor screening of AChE.

Prediction of Acute Cellular Renal Allograft Rejection by Urinary Metabolomics Using MALDI-FTMS

The present study investigated small molecule analysis of urinary samples as a noninvasive method to detect acute cellular renal allograft rejection. Matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) was used to analyze 15 urinary samples from transplant patients with different grades of biopsy showing improved clinical acute cellular rejection (ACR) and 24 urinary samples from 8 transplant patients without evidence of rejection. Seven small molecules demonstrated highly successful diagnostic performance (m/z): 278.1 (t = 3.398, p = 0.004), 293.0 (t = 2.169, p = 0.048), 294.1 (t = 2.154, p = 0.05), 382.2 (t = 2.961, p = 0.010), 383.3 (t = 2.270, p = 0.040), 402.2 (t = 2.994, p = 0.010), 424.0 (t = 2.644, p = 0.019). Kidney transplant patients with ACR could be distinguished from those without ACR using four individual small molecules with a specificity of 100%. In conclusion, the combination of MALDI-FTMS technology with a clear definition of patient groups can detect urine small molecule associated with ACR.

On the interaction of peptides with calcium ions as studied by matrix-assisted laser desorption/ionization Fourier transform mass spectrometry: Towards peptide fishing using metal ion baits

In a previous report [J.W. de Beukelaar, J.W. Gratama, P.A. Sillevis Smitt, G.M. Verjans, J. Kraan, Th.M. Luider, P.C. Burgers, Rapid Commun. Mass Spectrom. 21 (2007) 1282] on the quality assessment of synthetic peptides used in protein-spanning peptide pools by matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) we noted that certain peptides showed remarkably intense signals for their calcium-containing analogues. Here we report on a detailed mass spectrometric study of the unimolecular chemistry of these calcium-containing peptides. By integration of the experimental findings with computational results derived from DFT and the CBS-QB3 model chemistry, we have traced the processes induced by Ca 2+ attachment in the peptide ions. Key to our analysis is the observation that all of the studied calcium-bound peptides containing a threonine or serine residue show prominent losses of CH 3CH O (from threonine) and/or CH 2 O (from serine) in both the positive and the negative ion mode. In the first step, Ca 2+ attaches itself to a negatively charged in-chain carboxylate group. Next, electrophilic attack of the calcium ion on the CH(R)OH group of threonine (R CH 3) or serine (R H) releases the hydroxyl proton which can then move to a suitable acceptor site, viz. a peptide bond. This leads to the formation of a very stable ionic bidentate structure. Upon collisional activation (MS/MS), this bidentate opens up leading to the loss of the exposed acetaldehyde or formaldehyde molecule, to yield another bidentate structure. MS/MS spectra of selected peptides interacting with other metal ions have also been investigated and it is found that only divalent ions follow the Ca 2+-induced transformations.

SIFamide peptides in clawed lobsters and freshwater crayfish (Crustacea, Decapoda, Astacidea): A combined molecular, mass spectrometric and electrophysiological investigation

Recently, we identified the peptide VYRKPPFNGSIFamide (Val1-SIFamide) in the stomatogastric nervous system (STNS) of the American lobster Homarus americanus using matrix-assisted laser desorption/ionization-Fourier transform mass spectrometry (MALDI-FTMS). Given that H. americanus is the only species thus far shown to possess this peptide, and that a second SIFamide isoform, Gly1-SIFamide, is broadly conserved in other decapods, including another astacidean, the crayfish Procambarus clarkii, we became interested both in confirming our identification of Val1-SIFamide via molecular methods and in determining the extent to which this isoform is conserved within other members of the infraorder Astacidea. Here, we present the identification and characterization of an H. americanus prepro-SIFamide cDNA that encodes the Val1 isoform. Moreover, we demonstrate via MALDI-FTMS the presence of Val1-SIFamide in a second Homarus species, Homarus gammarus. In contrast, only the Gly1 isoform was detected in the other astacideans investigated, including the lobster Nephrops norvegicus, a member of the same family as Homarus, and the crayfish Cherax quadricarinatus, P. clarkii and Pacifastacus leniusculus, which represent members of each of the extant families of freshwater astacideans. These results suggest that Val1-SIFamide may be a genus (Homarus)-specific isoform. Interestingly, both Val1- and Gly1-SIFamide possess an internal dibasic site, Arg3-Lys4, raising the possibility of the ubiquitously conserved isoform PPFNGSIFamide. However, this octapeptide was not detected via MALDI-FTMS in any of the investigated species, and when applied to the isolated STNS of H. americanus possessed little bioactivity relative to the full-length Val1 isoform. Thus, it appears that the dodeca-variants Val1- and Gly1-SIFamide are the sole bioactive isoforms of this peptide family in clawed lobsters and freshwater crayfish.

Mass spectral characterization of peptide transmitters/hormones in the nervous system and neuroendocrine organs of the American lobster Homarus americanus

The American lobster Homarus americanus is a decapod crustacean with both high economic and scientific importance. To facilitate physiological investigations of peptide transmitter/hormone function in this species, we have used matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and nanoscale liquid chromatography coupled to electrospray ionization quadrupole time-of-flight tandem mass spectrometry (nanoLC-ESI-Q-TOF MS/MS) to elucidate the peptidome present in its nervous system and neuroendocrine organs. In total, 84 peptides were identified, including 27 previously known H. americanus peptides (e.g., VYRKPPFNGSIFamide [Val1-SIFamide]), 23 peptides characterized previously from other decapods, but new to the American lobster (e.g., pQTFQYSRGWTNamide [Arg7-corazonin]), and 34 new peptides de novo sequenced/detected for the first time in this study. Of particular note are a novel B-type allatostatin (TNWNKFQGSWamide) and several novel FMRFamide-related peptides, including an unsulfated analog of sulfakinin (GGGEYDDYGHLRFamide), two myosuppressins (QDLDHVFLRFamide and pQDLDHVFLRFamide), and a collection of short neuropeptide F isoforms (e.g., DTSTPALRLRFamide and FEPSLRLRFamide). Our data also include the first detection of multiple tachykinin-related peptides in a non-brachyuran decapod, as well as the identification of potential individual-specific variants of orcokinin and orcomyotropin-related peptide. Taken collectively, our results not only expand greatly the number of known H. americanus neuropeptides, but also provide a framework for future studies on the physiological roles played by these molecules in this commercially and scientifically important species.

Combining MALDI-FTMS and Bioinformatics for Rapid Peptidomic Comparisons

Increasing research efforts in large-scale mass spectral analyses of peptides and proteins have led to many advances in technology and method development for collecting data and improving the quality of data. However, the resultant large data sets often pose significant challenges in extracting useful information in a high-throughput manner. Here, we describe one such method where we analyzed a large mass spectral data set collected using decapod crustacean nervous tissue extracts separated via high-performance liquid chromatography (HPLC) coupled to high-resolution matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS). Following their acquisition, the data collected from discrete LC fractions was compiled and analyzed using an in-house developed software package that deisotoped, compressed, calibrated, and matched peaks to a list of known crustacean neuropeptides. By processing these data via bioinformatics tools such as hierarchical clustering, more than 110 neuropeptides that belong to 14 peptide families were mapped in five crustacean species. Overall, we demonstrate the utility of MALDI-FTMS in combination with a bioinformatics software package for the elucidation and comparison of peptidomes of varying crustacean species. This study established an effective methodology and will provide the basis for future investigations into more comprehensive comparative peptidomics with larger collection of species and phyla in order to gain a deeper understanding of the evolution and diversification of peptide families.

High-mass-resolution direct-tissue MALDI-FTMS reveals broad conservation of three neuropeptides (APSGFLGMRamide, GYRKPPFNGSIFamide and pQDLDHVFLRFamide) across members of seven decapod crustaean infraorders

Matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) has become an important method for identifying peptides in neural tissues. The ultra-high-mass resolution and mass accuracy of MALDI-FTMS, in combination with in-cell accumulation techniques, can be used to advantage for the analysis of complex mixtures of peptides directly from tissue fragments or extracts. Given the diversity within the decapods, as well as the large number of extant species readily available for analysis, this group of animals represents an optimal model in which to examine phylogenetic conservation and evolution of neuropeptides and neuropeptide families. Surprisingly, no large comparative studies have previously been undertaken. Here, we have initiated such an investigation, which encompasses 32 species spanning seven decapod infraorders. Two peptides, APSGFLGMRamide and pQDLDHVFLRFamide, were detected in all species. A third peptide, GYRKPPFNGSIFamide, was detected in all species except members of the Astacidean genus Homarus, where a Val 1 variant was present. Our finding that these peptides are ubiquitously (or nearly ubiquitously) conserved in decapod neural tissues not only suggests important conserved functions for them, but also provides an intrinsic calibrant set for future MALDI-FTMS assessments of other peptides in this crustacean order.

Direct tissue MALDI-FTMS profiling of individual Cancer productus sinus glands reveals that one of three distinct combinations of crustacean hyperglycemic hormone precursor-related peptide (CPRP) isoforms are present in individual crabs

Over the past decade, mass spectrometry has become a prominent technique for identifying peptide hormones. In crustaceans, studies directed at characterizing the peptide complements present in neuroendocrine structures have generally involved the isolation of tissue from a large number of individuals, which are pooled, extracted, purified, and then analyzed via chromatographic techniques coupled with mass spectrometry. While this approach provides information on the peptides present in the population of animals used as the tissue source, data on the peptide complement present in any individual animal are lost. Direct tissue matrix assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) of single tissues has the potential to identify differences in peptide expression between individuals. Here, we have used direct tissue MALDI-FTMS of individual sinus glands (SGs) to show that the four isoforms of crustacean hyperglycemic hormone precursor-related peptide (CPRP) identified previously from pooled Cancer productus SGs ( i.e. Fu, Q., Christie, A.E., Li, L. 2005. Mass spectrometric characterization of crustacean hyperglycemic hormone precursor-related peptides (CPRPs) from the sinus gland of the crab, Cancer productus. Peptides 26, 2137–2150.) are differentially distributed in conserved patterns among individual crabs. Of the crabs examined, ∼61% of the individuals possessed Capr-CPRP I and II, but not III or IV, ∼26% Capr-CPRP I, II and III, but not IV, and ∼13% Capr-CPRP I, II and IV, but not III. Our findings set the stage for future molecular investigations on the origin(s) of this individual-specific variation in CPRP complement, as well as investigations of the function and regulation of the individual isoforms. These data also lend a cautionary note to the assumption that the peptides identified via pooled tissues reveal an accurate picture of the peptides present in any given individual.