New developments in profiling and imaging of proteins from tissue sections by MALDI mass spectrometry

Molecular imaging of tissue by MALDI mass spectrometry is a powerful tool for visualizing the spatial distribution of constituent analytes with high molecular specificity. Although the technique is relatively young, it has already contributed to the understanding of many diverse areas of human health. In recent years, a great many advances in the practice of imaging mass spectrometry have taken place, making the technique more sensitive, robust, and ultimately useful. The purpose of this review is to highlight some of the more recent technological advances that have improved the efficiency of imaging mass spectrometry for clinical applications. Advances in the way MALDI mass spectrometry is integrated with histology, improved methods for automation, and better tools for data analysis are outlined in this review. Refined top-down strategies for the identification and validation of candidate biomarkers found in tissue sections are discussed. A clinical example highlighting the application of these methods to a cohort of clinical samples is described.     

Proteomic analysis of cast cuticles from Anopheles gambiae by tandem mass spectrometry

Identification of authenticated cuticular proteins has been based on isolation and sequencing of individual proteins extracted from cleaned cuticles. These data facilitated classification of sequences from conceptual translation of cDNA or genomic sequences. The question arises whether such putative cuticular proteins actually are incorporated into the cuticle. This paper describes the profiling of cuticular proteins from Anopheles gambiae starting with cuticle cleaned by the insect itself in the course of molting. Proteins extracted from cast larval head capsules and cast pupal cuticles were fractionated by 1D SDS gel electrophoresis. Large gel slices were reduced, carbamidomethylated and digested with trypsin. The pellet remaining after SDS extraction was also treated with trypsin. The resulting peptides were separated on a C18 column and then analyzed by tandem mass spectrometry. Two-hundred-ninety-five peptides from putative cuticular proteins were identified; these corresponded to a minimum of 69 and a maximum of 119 different proteins. Each is reported as an authentic Anopheles cuticular protein for the first time. In addition to members of two known cuticular protein families, members of additional families likely to be structural components of the cuticle were identified. Furthermore, other peptides were identified that can be attributed to molting fluid, muscle and sclerotizing agents.     

A simple desalting method for direct MALDI mass spectrometry profiling of tissue lipids

Direct MALDI-mass spectrometry (MALDI-MS) profiling of tissue lipids often observes isobaric phosphatidylcholine (PC) species caused by the endogenous alkali metal ions that bias the relative abundance of tissue lipids. Fresh rat brain cryosections were washed with 70% etha­nol (EtOH), water (H₂O), or 150 mM ammonium acetate (NH₄Ac), and the desalting effectiveness of each fluid was evaluated by MALDI-MS profiling of PC and sphingomyelin (SM) species in tissue and in the washing runoff. The results indicated that EtOH and H₂O only partially desalted the tissue lipids, yet both substantially displaced the tissue lipids to the washing runoffs. On the other hand, NH₄Ac effectively desalted the tissue lipids and produced a runoff containing no detectable PCs or SMs. NH₄Ac wash also unveiled the underlying changes of PCs and SMs in the infarcted rat cortex previously masked by edema-caused increase of tissue sodium. The MS/MS of an isobaric PC in the infarcted cortex revealed the precursor change as the result of NH₄Ac wash and confirmed the desalting effectiveness of such wash. Other than desalting, NH₄Ac wash also removes contaminants in tissue, enhances the overall spectral quality, and benefits additionally in profiling of biological molecules in tissue.     

The MALDI mass spectrometry in the identification of new proteins in snake venoms

By MALDI MS, we searched cobra venoms for new low-content polypeptides. A number of new proteins with molecular masses 7–25 kDa, characteristic of the known snake protein toxins, were identified, with the content of one of them less than 0.02%.     

Sex-biased expression of odorant receptors in antennae and palps of the African malaria vector Anopheles gambiae

At the heart of the odor recognition process in all animals are G-protein-coupled receptors, which are seven-transmembrane domain proteins that initiate G-protein-mediated signaling cascades when activated by their ligands. Odorant receptors (ORs) are a large, diverse family of proteins with some 80 members in the mosquito Anopheles gambiae. With the assumption that more sensilla on female antennae are tuned to human odors than on male antennae, comparison of specific OR mRNA levels in male and female antennae can provide an indication as to which receptors may be stimulated by host odors. We have used RT PCR and quantitative real-time PCR (qRT PCR) to investigate sex-biased expression levels of 80 A. gambiae ORs in male and female antennae and maxillary palps. On the basis of prevalence of expression in female antennae and on a strong female relative to male expression bias we identified a short list of ORs that are likely involved in host odor recognition by female mosquitoes.     

Identification and expression profiling of putative odorant-binding proteins in the malaria mosquitoes, Anopheles gambiae and A.arabiensis

~~Identification and expression profiling of putative odorant-binding proteins in the malaria mosquitoes, Anopheles gambiae and A. arabiensis1. Curtis, C. F., Introduction 1: An overview of mosquito biology, behaviour and importance, in Olfaction in Mosquito-Host Interactions (eds. Bock, G. R.. Cardew, G.), New York: Wiley, 1996, 3-7. 2. Nighom, A., Hildebrand. J. G.. Dissecting the molecular mechanisms of olfaction in a malaria-vector mosquito, PNAS, 2002, 99(3): 1113-…     

Molecular profiling of experimental Parkinson's disease: direct analysis of peptides and proteins on brain tissue sections by MALDI mass spectrometry

Direct molecular profiling of biological samples using matrix-assisted laser desorption ionization mass spectrometry is a powerful tool for identifying phenotypic markers. In this report, protein profiling was used for the first time to generate peptide and protein profiles of brain tissue sections obtained from experimental Parkinson's disease (unilaterally 6-hydroxydopamine treated rats). The mass spectrometer was used to map the peptide and protein expression directly on 12 microm tissue sections in mass-to-charge (m/z) values, providing the capability of mapping specific molecules of the original sample, that is, localization, intensity and m/z ratio. Several protein expression profile differences were found in the dopamine depleted side of the brain when compared to the corresponding intact side, for example, calmodulin, cytochrome c, and cytochrome c oxidase. An increased ratio of post-translational modifications such as acetylations were found in the striatum of proteins in the dopamine depleted side of the brain. These modifications were decreased after subchronic administration of L-Dopa. The present study shows that unique protein profiles can be obtained in specific brain regions (and subregions) directly on brain tissue sections and allows for the study of complex biochemical processes such as those occurring in experimental Parkinson's disease.     

Strain differentiation of Staphylococcus aureus by means of direct MALDI TOF mass spectrometry profiling

Staphylococcus aureus is one of the most interesting microbial species in clinical studies. It is characterized by a wide extent of strain diversity, first of all, due to variability in virulence and pathogenicity. The aim of this study was to test the method of rapid Staphylococcus strain differentiation by a certain sign based on registration of characteristics features of MALDI mass spectra accumulated during direct protein profiling of the bacterial cell. The model signs registered as strain differences included production of β-lactamase and α-hemolysin encoded by blaZ and hla genes, respectively. The mathematical analysis of MALDI mass spectra accumulated for 53 S. aureus isolates using the clustering genetic algorithm resulted in generation of two independent classification models, which could differentiate the strains by the considered features. Using statistical contribution of each mass peak to the model, the most significant peaks (masses), which could be considered as the markers of Staphylococcus strain differences, were found. The generated diagnostic models were characterized by the following sensitivity and specificity coefficients: 97.5 and 82.5%, respectively, for strain differentiation by β-lactamase production and 90.0 and 88.7% by the presence of α-hemolysin.     

Studies on the bionomics of male Anopheles gambiae Giles and male Anopheles funestus Giles from southern Mozambique

Little is known about the fitness of wild male mosquitoes, the females of which are vectors of malaria. The problem of studying male biology has been exacerbated by difficulties associated with catching them. In southern Mozambique, however, almost the entire adult population of An. funestus and An. gambiae s.l. rest inside houses. They leave in a dusk exodus, which makes them easy to collect. In 8,348 exit collections from a village from 2003 to 2009, 567,195 male An. funestus and 34,591 male An. gambiae s.l. were collected. During the study, numbers of An. funestus increased but numbers of An. gambiae s.l. declined to the point of extinction. Overall numbers of An. gambiae s.l. were positively correlated with temperature, whilst the relationship between temperature and numbers of An. funestus changed from an initially positive one in the first three years of the study to a negative one in the last three years. Marked males were recaptured up to 300 m from the release site, with most recaptures occurring within 150 m. Estimated mean daily survival of male An. funestus was 0.86 (95% C.I. 0.869-0.850). For the years 2003-2007, estimated mean daily survival of male An gambiae s.l. was 0.660 (95% C.I. 0.682-0.638). For either species, there was no relationship between mean weekly temperature and estimated daily survival. These results imply that males of An. funestus live as long as females but have a relatively short flight range. They are discussed in the light of possible release strategies of sterile or genetically modified mosquitoes.     

冈比亚按蚊嗅觉结合蛋白候选基因cDNA的克隆、鉴定及其表达型分析

疟蚊主要依靠嗅觉发现寄主。非洲疟蚊冈比亚按蚊Anopheles gambiae是一种嗜吸人血的疟疾传播媒介昆虫。该文作者基于其全基因组序列,采用RT-PCR和标准分子克隆技术获得2个嗅觉结合蛋白候选基因agLZ3788和agLZ9988。测序分析结果表明,它们具有嗅觉结合蛋白的标志性结构域。进一步采用半定量RT-PCR技术研究了它们的空间表达型,结果发现它们不但在雌蚊触角中表达,也在其他部位(尤其是蚊虫足部)有强的表达。这一发现说明疟蚊嗅觉结合蛋白可能具有更广的功能,也为进一步重组表达和功能研究提供了重要依据。     

Sample preparation and bioinformatics in MALDI profiling of urinary proteins

One of the challenges of current proteomics research is identifying healthy and diseased mass spectrometric (MS) patterns within biological fluids. As a result, sample preparation methodologies, as well as the mathematical tools utilized for MS data analysis become pivotal. This study involves a thorough evaluation of the reproducibility and protein resolution that various urinary protein preparation methodologies provide in MALDI MS analysis. Several precipitation approaches, ultrafiltration, as well as direct dilution of urine in MALDI MS compatible buffers were applied in combination to a thorough bioinformatics analysis of the generated MS data. Our results indicate that ultrafiltration, as well as direct dilution of urine in TFA, can provide information rich and reproducible spectra for mass ranges up to 20 kDa. The importance of the presence of peak reproducibility filters when generating disease classification models is suggested.     

Identification and expression of odorant-binding proteins of the malaria-carrying mosquitoes Anopheles gambiae and Anopheles arabiensis

Host preference and blood feeding are restricted to female mosquitoes. Olfaction plays a major role in host-seeking behaviour, which is likely to be associated with a subset of mosquito olfactory genes. Proteins involved in olfaction include the odorant receptors (ORs) and the odorant-binding proteins (OBPs). OBPs are thought to function as a carrier within insect antennae for transporting odours to the olfactory receptors. Here we report the annotation of 32 genes encoding putative OBPs in the malaria mosquito Anopheles gambiae and their tissue-specific expression in two mosquito species of the Anopheles complex; a highly anthropophilic species An. gambiae sensu stricto and an opportunistic, but more zoophilic species, An. arabiensis. RT-PCR shows that some of the genes are expressed mainly in head tissue and a subset of these show highest expression in female heads. One of the genes (agCP1588) which has not been identified as an OBP, has a high similarity (40%) to the Drosophila pheromone-binding protein 4 (PBPRP4) and is only expressed in heads of both An. gambiae and An. arabiensis, and at higher levels in female heads. Two genes (agCP3071 and agCP15554) are expressed only in female heads and agC15554 also shows higher expression levels in An. gambiae. The expression profiles of the genes in the two members of the Anopheles complex provides the first step towards further molecular analysis of the mosquito olfactory apparatus.     

MALDI mass spectrometry in prostate cancer biomarker discovery

Matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry (MS) is a highly versatile and sensitive analytical technique, which is known for its soft ionisation of biomolecules such as peptides and proteins. Generally, MALDI MS analysis requires little sample preparation, and in some cases like MS profiling it can be automated through the use of robotic liquid-handling systems. For more than a decade now, MALDI MS has been extensively utilised in the search for biomarkers that could aid clinicians in diagnosis, prognosis, and treatment decision making. This review examines the various MALDI-based MS techniques like MS imaging, MS profiling and proteomics in-depth analysis where MALDI MS follows fractionation and separation methods such as gel electrophoresis, and how these have contributed to prostate cancer biomarker research. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.     

Distribution of cuticular proteins in different structures of adult Anopheles gambiae

Anopheles gambiae devotes over 2% (295) of its protein coding genes to structural cuticular proteins (CPs) that have been classified into 13 different families plus ten low complexity proteins not assigned to families. Small groups of genes code for identical proteins reducing the total number of unique cuticular proteins to 282. Is the large number because different structures utilize different CPs, or are all of the genes widely expressed? We used LC-MS/MS to learn how many products of these genes were found in five adult structures: Johnston's organs, the remainder of the male antennae, eye lenses, legs, and wings. Data were analyzed against both the entire proteome and a smaller database of just CPs. We recovered unique peptides for 97 CPs and shared peptides for another 35. Members of 11 of the 13 families were recovered as well as some unclassified. Only 11 CPs were present exclusively in only one structure while 43 CPs were recovered from all five structures. A quantitative analysis, using normalized spectral counts, revealed that only a few CPs were abundant in each structure. When the MS/MS data were run against the entire proteome, the majority of the top hits were to CPs, but peptides were recovered from an additional 467 proteins. CP peptides were frequently recovered from chitin-binding domains, confirming that protein-chitin interactions are not mediated by covalent bonds. Comparison with three other MS/MS analyses of cuticles or cuticle-rich structures augmented the current analysis. Our findings provide new insights into the composition of different mosquito structures and reveal the complexity of selection and utilization of genes coding for structural cuticular proteins.     

Quantitative profiling of proteins in complex mixtures using liquid chromatography and mass spectrometry

The objective of this study was to determine if liquid chromatography mass spectrometry (LC/MS) data of tryptic digests of proteins can be used for quantitation. In theory, the peak area of peptides should correlate to their concentration; hence, the peak areas of peptides from one protein should correlate to the concentration of that particular protein. To evaluate this hypothesis, different amounts of tryptic digests of myoglobin were analyzed by LC/MS in a wide range between 10 fmol and 100 pmol. The results show that the peak areas from liquid chromatography mass spectrometry correlate linearly to the concentration of the protein (r2 = 0.991). The method was further evaluated by adding two different concentrations of horse myoglobin to human serum. The results confirm that the quantitation method can also be used for quantitative profiling of proteins in complex mixtures such as human sera. Expected and calculated protein ratios differ by no more than 16%. We describe a new method combining protein identification with accurate profiling of individual proteins. This approach should provide a widely applicable means to compare global protein expression in biological samples.     

Bioinformatics-based identification of chemosensory proteins in African Malaria Mosquito, Anopheles gambiae

Chemosensory proteins (CSPs) are identifiable by four spatially conserved Cys-teine residues in their primary structure or by two disulfide bridges in their tertiary structure according to the previously identified olfactory specific-D related proteins. A genomics- and bioinformatics-based approach is taken in the present study to identify the putative CSPs in the malaria-carrying mosquito, Anopheles     

Switchover to the mated state by spermathecal activation in female Anopheles gambiae mosquitoes

Both Aedes aegypti and Anopheles gambiae mosquitoes undergo physiological and behavioral changes after the females mate, but unlike Ae. aegypti, the mated state in An. gambiae is not attained through the action of male accessory gland substances. Experiments in which the spermathecae of mated females were manipulated suggest that a spermatheca filled with sperm is responsible for triggering oviposition behavior. An estimated 48% of the previously mated An. gambiae females mated again when the interval between encounters with males was less than 24h. Unlike male Ae. aegypti that can inseminate up to seven females after their testes are removed, An. gambiae do not store sperm in the vas deferens and without testes would be unable to inseminate any females.     

Site-specific phosphorylation profiling of Arabidopsis proteins by mass spectrometry and peptide chip analysis

An estimated one-third of all proteins in higher eukaryotes are regulated by phosphorylation by protein kinases (PKs). Although plant genomes encode more than 1000 PKs, the substrates of only a small fraction of these kinases are known. By mass spectrometry of peptides from cytoplasmic- and nuclear-enriched fractions, we determined 303 in vivo phosphorylation sites in Arabidopsis proteins. Among 21 different PKs, 12 were phosphorylated in their activation loops, suggesting that they were in their active state. Immunoblotting and mutational analysis confirmed a tyrosine phosphorylation site in the activation loop of a GSK3/shaggy-like kinase. Analysis of phosphorylation motifs in the substrates suggested links between several of these PKs and many target sites. To perform quantitative phosphorylation analysis, peptide arrays were generated with peptides corresponding to in vivo phosphorylation sites. These peptide chips were used for kinome profiling of subcellular fractions as well as H 2O 2-treated Arabidopsis cells. Different peptide phosphorylation profiles indicated the presence of overlapping but distinct PK activities in cytosolic and nuclear compartments. Among different H 2O 2-induced PK targets, a peptide of the serine/arginine-rich (SR) splicing factor SCL30 was most strongly affected. SRPK4 (SR protein-specific kinase 4) and MAPKs (mitogen-activated PKs) were found to phosphorylate this peptide, as well as full-length SCL30. However, whereas SRPK4 was constitutively active, MAPKs were activated by H 2O 2. These results suggest that SCL30 is targeted by different PKs. Together, our data demonstrate that a combination of mass spectrometry with peptide chip phosphorylation profiling has a great potential to unravel phosphoproteome dynamics and to identify PK substrates.     

Genetic variation of male reproductive success in a laboratory population of Anopheles gambiae

Background

A previous study showed for Anopheles gambiae s.s. a gradation of feeding preference on common plant species growing in a malaria holoendemic area in western Kenya. The present follow-up study determines whether there is a relationship between the mosquito's preferences and its survival and fecundity.

Methods

Groups of mosquitoes were separately given ad libitum opportunity to feed on five of the more preferred plant species (Hamelia patens, Parthenium hysterophorus, Ricinus communis, Senna didymobotrya, and Tecoma stans) and one of the less preferred species (Lantana camara). The mosquitoes were monitored daily for survival. Sugar solution (glucose 6%) and water were used as controls. In addition, the fecundity of mosquitoes on each plant after (i) only one blood meal (number of eggs oviposited), and (ii) after three consecutive blood meals (proportion of females ovipositing, number of eggs oviposited and hatchability of eggs), was determined. The composition and concentration of sugar in the fed-on parts of each plant species were determined using gas chromatography. Using SAS statistical package, tests for significant difference of the fitness values between mosquitoes exposed to different plant species were conducted.

Results and Conclusion

Anopheles gambiae that had fed on four of the five more preferred plant species (T. stans, S. didymobotrya, R. communis and H. patens, but not P. hysterophorus) lived longer and laid more eggs after one blood meal, when compared with An. gambiae that had fed on the least preferred plant species L. camara. When given three consecutive blood-meals, the percentage of females that oviposited, but not the number of eggs laid, was significantly higher for mosquitoes that had previously fed on the four more preferred plant species. Total sugar concentration in the preferred plant parts was significantly correlated with survival and with the proportion of females that laid eggs. This effect was associated mainly with three sugar types, namely glucose, fructose, and gulose. Except for P. hysterophorus, the results suggest that feeding by mosquitoes on preferred plant species under natural conditions results in higher fitness-related benefits, and that the sugar content in preferred plant parts is largely responsible for these effects.