Comparison of metaheuristic strategies for peakbin selection in proteomic mass spectrometry data

Mass spectrometry (MS) data provide a promising strategy for biomarker discovery. For this purpose, the detection of relevant peakbins in MS data is currently under intense research. Data from mass spectrometry are challenging to analyze because of their high dimensionality and the generally low number of samples available. To tackle this problem, the scientific community is becoming increasingly interested in applying feature subset selection techniques based on specialized machine learning algorithms. In this paper, we present a performance comparison of some metaheuristics: best first (BF), genetic algorithm (GA), scatter search (SS) and variable neighborhood search (VNS). Up to now, all the algorithms, except for GA, have been first applied to detect relevant peakbins in MS data. All these metaheuristic searches are embedded in two different filter and wrapper schemes coupled with Naive Bayes and SVM classifiers.

Double ion implantation and pulsed laser melting processes for third generation solar cells

In the framework of the third generation of photovoltaic devices, the intermediate band solar cell is one of the possible candidates to reach higher efficiencies with a lower processing cost. In this work, we introduce a novel processing method based on a double ion implantation and, subsequently, a pulsed laser melting (PLM) process to obtain thicker layers of Ti supersaturated Si. We perform ab initio theoretical calculations of Si impurified with Ti showing that Ti in Si is a good candidate to theoretically form an intermediate band material in the Ti supersaturated Si. From time-of-flight secondary ion mass spectroscopy measurements, we confirm that we have obtained a Ti implanted and PLM thicker layer of 135 nm. Transmission electron microscopy reveals a single crystalline structure whilst the electrical characterization confirms the transport properties of an intermediate band material/Si substrate junction. High subbandgap absorption has been measured, obtaining an approximate value of 104 cm−1 in the photons energy range from 1.1 to 0.6 eV.

Plasma dynamics studies by mass spectrometry techniques

We present temporal information obtained by mass spectrometry techniques about the evolution of plasmas generated by laser filamentation in air. The experimental setup used in this work allowed us to study not only the dynamics of the filament core but also of the energy reservoir that surrounds it. Furthermore, valuable insights about the chemistry of such systems like the photofragmentation and/or formation of molecules were obtained. The interpretation of the experimental results are supported by PIC simulations.

Multiresidue Analysis of Insecticides and Other Selected Environmental Contaminants in Poultry Manure by Gas Chromatography/Mass Spectrometry

An analytical method was developed for the simultaneous determination in poultry manure of 41 organic contaminants belonging to different chemical classes: pesticides, polycyclic aromatic hydrocarbons, polychlorinated biphenyls, and polybrominated diphenyl ethers. Poultry manure was extracted with a modified QuEChERS method, and the extracts were analyzed by isotope dilution GC/MS. Recovery of these contaminants from samples spiked at levels ranging from 25 to 100 ng/g was satisfactory for all the compounds. The developed procedure provided LODs from 0.8 to 9.6 ng/g. The analysis of poultry manure samples collected on different farms confirmed the presence of some of the studied contaminants. Pyrethroids and polycyclic aromatic hydrocarbons were the main contaminants detected.

Identification by mass spectrometry of the phosphorylated residue responsible for activation of the catalytic domain of myosin I heavy chain kinase, a member of the PAK/STE20 family

Myosin I heavy chain kinase from Acanthamoeba castellanii is activated in vitro by autophosphorylation (8–10 mol of P per mol). The catalytically active C-terminal domain produced by trypsin cleavage of the phosphorylated kinase contains 2–3 mol of P per mol. However, the catalytic domain expressed in a baculovirus–insect cell system is fully active as isolated without autophosphorylation in vitro. We now show that the expressed catalytic domain is inactivated by incubation with acid phosphatase and regains activity upon autophosphorylation. The state of phosphorylation of all of the hydroxyamino acids in the catalytic domain were determined by mass spectrometry of unfractionated protease digests. Ser-627 was phosphorylated in the active, expressed catalytic domain, lost its phosphate when the protein was incubated with phosphatase, and was rephosphorylated when the dephosphorylated protein was incubated with ATP. No other residue was significantly phosphorylated in any of the three samples. Thus, phosphorylation of Ser-627, which is in the same position as the Ser and Thr residues that are phosphorylated in many other kinases, is necessary and sufficient for full activity of the catalytic domain. Ser-627 is also phosphorylated when full-length, native kinase is activated by autophosphorylation.

Linking genome and proteome by mass spectrometry: Large-scale identification of yeast proteins from two dimensional gels

The function of many of the uncharacterized open reading frames discovered by genomic sequencing can be determined at the level of expressed gene products, the proteome. However, identifying the cognate gene from minute amounts of protein has been one of the major problems in molecular biology. Using yeast as an example, we demonstrate here that mass spectrometric protein identification is a general solution to this problem given a completely sequenced genome. As a first screen, our strategy uses automated laser desorption ionization mass spectrometry of the peptide mixtures produced by in-gel tryptic digestion of a protein. Up to 90% of proteins are identified by searching sequence data bases by lists of peptide masses obtained with high accuracy. The remaining proteins are identified by partially sequencing several peptides of the unseparated mixture by nanoelectrospray tandem mass spectrometry followed by data base searching with multiple peptide sequence tags. In blind trials, the method led to unambiguous identification in all cases. In the largest individual protein identification project to date, a total of 150 gel spots—many of them at subpicomole amounts—were successfully analyzed, greatly enlarging a yeast two-dimensional gel data base. More than 32 proteins were novel and matched to previously uncharacterized open reading frames in the yeast genome. This study establishes that mass spectrometry provides the required throughput, the certainty of identification, and the general applicability to serve as the method of choice to connect genome and proteome.

Analysis of receptor signaling pathways by mass spectrometry: Identification of Vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors

Oligomerization of receptor protein tyrosine kinases such as the epidermal growth factor receptor (EGFR) by their cognate ligands leads to activation of the receptor. Transphosphorylation of the receptor subunits is followed by the recruitment of signaling molecules containing src homology 2 (SH2) or phosphotyrosine interaction domains (PID). Additionally, several cytoplasmic proteins that may or may not associate with the receptor undergo tyrosine phosphorylation. To identify several components of the EGFR signaling pathway in a single step, we have immunoprecipitated molecules that are tyrosine phosphorylated in response to EGF and analyzed them by one-dimensional gel electrophoresis followed by mass spectrometry. Combining matrix-assisted laser desorption/ionization (MALDI) and nanoelectrospray tandem mass spectrometry (MS/MS) led to the identification of nine signaling molecules, seven of which had previously been implicated in EGFR signaling. Several of these molecules were identified from low femtomole levels of protein loaded onto the gel. We identified Vav-2, a recently discovered guanosine nucleotide exchange factor that is expressed ubiquitously, as a substrate of the EGFR. We demonstrate that Vav-2 is phosphorylated on tyrosine residues in response to EGF and associates with the EGFR in vivo. Binding of Vav-2 to the EGFR is mediated by the SH2 domain of Vav-2. In keeping with its ubiquitous expression, Vav-2 seems to be a general signaling molecule, since it also associates with the platelet-derived growth factor (PDGF) receptor and undergoes tyrosine phosphorylation in fibroblasts upon PDGF stimulation. The strategy suggested here can be used for routine identification of downstream components of cell surface receptors in mammalian cells.

Direct genetic analysis by matrix-assisted laser desorption/ionization mass spectrometry

An approach to analyzing single-nucleotide polymorphisms (SNPs) found in the human genome has been developed that couples a recently developed invasive cleavage assay for nucleic acids with detection by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The invasive cleavage assay is a signal amplification method that enables the analysis of SNPs by MALDI-TOF MS directly from human genomic DNA without the need for initial target amplification by PCR. The results presented here show the successful genotyping by this approach of twelve SNPs located randomly throughout the human genome. Conventional Sanger sequencing of these SNP positions confirmed the accuracy of the MALDI-TOF MS analysis results. The ability to unambiguously detect both homozygous and heterozygous genotypes is clearly demonstrated. The elimination of the need for target amplification by PCR, combined with the inherently rapid and accurate nature of detection by MALDI-TOF MS, gives this approach unique and significant advantages in the high-throughput genotyping of large numbers of SNPs, useful for locating, identifying, and characterizing the function of specific genes.

Metabolism of an insect diuretic hormone by Malpighian tubules studied by liquid chromatography coupled with electrospray ionization mass spectrometry

The larger of two diuretic hormones of the tobacco hornworm, Manduca sexta, (Mas-DH) is a peptide of 41 residues. It is one of a family of seven currently known insect diuretic hormones that are similar to the corticotropin-releasing factor–urotensin–sauvagine family of peptides. We investigated the possible inactivation of Mas-DH by incubating it in vitro with larval Malpighian tubules (Mt), the target organ of the hormone. The medium was analyzed, and degradation products were identified, using on-line microbore reversed-phase liquid chromatography coupled to electrospray ionization mass spectrometry (RPLC-ESI-MS). This sensitive technique allows identification of metabolites of Mas-DH (present at an initial level of ≈1 μM). An accurate Mr value for a metabolite is usually sufficient for unambiguous identification. Mas-DH is cleaved by Mt proteases initially at L29–R30 and R30–A31 under our assay conditions; some Mas-DH is also oxidized, apparently at M2 and M11. The proteolysis can be inhibited by 5 mM EDTA, suggesting that divalent metals are needed for peptide cleavage. The oxidation of the hormone can be inhibited by catalase or 1 mM methionine, indicating that H2O2 or related reactive oxygen species are responsible for the oxidative degradation observed. RPLC-ESI-MS is shown here to be an elegant and efficient method for studying peptide hormone metabolism resulting from unknown proteases and pathways.

The earliest archaeological maize (Zea mays L.) from highland Mexico: New accelerator mass spectrometry dates and their implications

Accelerator mass spectrometry age determinations of maize cobs (Zea mays L.) from Guilá Naquitz Cave in Oaxaca, Mexico, produced dates of 5,400 carbon-14 years before the present (about 6,250 calendar years ago), making those cobs the oldest in the Americas. Macrofossils and phytoliths characteristic of wild and domesticated Zea fruits are absent from older strata from the site, although Zea pollen has previously been identified from those levels. These results, together with the modern geographical distribution of wild Zea mays, suggest that the cultural practices that led to Zea domestication probably occurred elsewhere in Mexico. Guilá Naquitz Cave has now yielded the earliest macrofossil evidence for the domestication of two major American crop plants, squash (Cucurbita pepo) and maize.

DNA-bound transcription factor complexes analysed by mass-spectrometry: binding of novel proteins to the human c-fos SRE and related sequences

Transcription factors control eukaryotic polymerase II function by influencing the recruitment of multiprotein complexes to promoters and their subsequent integrated function. The complexity of the functional ‘transcriptosome’ has necessitated biochemical fractionation and subsequent protein sequencing on a grand scale to identify individual components. As a consequence, much is now known of the basal transcription complex. In contrast, less is known about the complexes formed at distal promoter elements. The c-fos SRE, for example, is known to bind Serum Response Factor (SRF) and ternary complex factors such as Elk-1. Their interaction with other factors at the SRE is implied but, to date, none have been identified. Here we describe the use of mass-spectrometric sequencing to identify six proteins, SRF, Elk-1 and four novel proteins, captured on SRE duplexes linked to magnetic beads. This approach is generally applicable to the characterisation of nucleic acid-bound protein complexes and the post-translational modification of their components.

DNA sequencing and genotyping by transcriptional synthesis of chain-terminated RNA ladders and MALDI-TOF mass spectrometry

Sets of RNA ladders can be synthesized by transcription of a bacteriophage-encoded RNA polymerase using 3′-deoxynucleotides as chain terminators. These ladders can be used for sequencing of DNA. Using a nicked form of phage SP6 RNA polymerase in this study substantially enhanced yields of transcriptional sequencing ladders. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) of chain-terminated RNA ladders allowed DNA sequence determination of up to 56 nt. It is also demonstrated that A→G and C→T variations in heterozygous and homozygous samples can be unambiguously identified by the mass spectrometric analysis. As a step towards single-tube sequencing reactions, α-thiotriphosphate nucleotide analogs were used to overcome problems caused by chain terminator-independent, premature termination and by the small mass difference between natural pyrimidine nucleotides.

Attomole level protein sequencing by Edman degradation coupled with accelerator mass spectrometry

Edman degradation remains the primary method for determining the sequence of proteins. In this study, accelerator mass spectrometry was used to determine the N-terminal sequence of glutathione S-transferase at the attomole level with zeptomole precision using a tracer of 14C. The transgenic transferase was labeled by growing transformed Escherichia coli on [14C]glucose and purified by microaffinity chromatography. An internal standard of peptides on a solid phase synthesized to release approximately equal amounts of all known amino acids with each cycle were found to increase yield of gas phase sequencing reactions and subsequent semimicrobore HPLC as did a lactoglobulin carrier. This method is applicable to the sequencing of proteins from cell culture and illustrates a path to more general methods for determining N-terminal sequences with high sensitivity.