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Lloyd M Smith

Publications and source records attributed to Lloyd M Smith.

At least 19 recordsLinked to original sources
PubMed2025

IsoBayes: a Bayesian approach for single-isoform proteomics inference.

MOTIVATION: Studying protein isoforms is an essential step in biomedical research; at present, the main approach for analyzing proteins is via bottom-up mass spectrometry proteomics, which return peptide identifications, that are indirectly used to infer the presence of protein isoforms. However, the detection and quantification processes are noisy; in particular, peptides may be erroneously detected, and most peptides, known as shared peptides, are associated to multiple protein isoforms. As a consequence, studying individual protein isoforms is challenging, and inferred protein results are often abstracted to the gene-level or to groups of protein isoforms. RESULTS: Here, we introduce IsoBayes, a novel statistical method to perform inference at the isoform level. Our method enhances the information available, by integrating mass spectrometry proteomics and transcriptomics data in a Bayesian probabilistic framework. To account for the uncertainty in the measurement process, we propose a two-layer latent variable approach: first, we sample if a peptide has been correctly detected (or, alternatively filter peptides); second, we allocate the abundance of such selected peptides across the protein(s) they are compatible with. This enables us, starting from peptide-level data, to recover protein-level data; in particular, we: (i) infer the presence/absence of each protein isoform (via a posterior probability), (ii) estimate its abundance (and credible interval), and (iii) target isoforms where transcript and protein relative abundances significantly differ. We benchmarked our approach in simulations, and in two multi-protease real datasets: our method displays good sensitivity and specificity when detecting protein isoforms, its estimated abundances highly correlate with the ground truth, and can detect changes between protein and transcript relative abundances. AVAILABILITY AND IMPLEMENTATION: IsoBayes is freely distributed as a Bioconductor R package, and is accompanied by an example usage vignette.

Proteomics↗
PubMed2025

Improved Detection of Differentially Abundant Proteins through FDR-Control of Peptide-Identity-Propagation.

The goal of proteomics is to identify and quantify peptides and proteins within a biological sample. Almost all algorithms for the identification of peptides in LC-MS/MS data employ two steps: peptide/spectrum matching and peptide-identity-propagation (PIP), also known as match-between-runs. PIP can routinely account for up to 40% of all results, with that proportion rising as high as 75% in single-cell proteomics. Unlike peptide identities derived through peptide/spectrum matches, for which error estimation has been strictly enforced for decades, peptide identities derived through PIP have not historically been subject to statistical evaluation. As an indispensable component of label-free quantification, PIP needs a statistically rigorous method for estimating its false-discovery rate (FDR). We present a method for FDR control of PIP, called PIP-ECHO, and devise a rigorous protocol for evaluating FDR control of any PIP method. Using three different benchmark data sets, we evaluate PIP-ECHO alongside the PIP procedures implemented by FlashLFQ, IonQuant, and MaxQuant. These analyses show that only PIP-ECHO can accurately control the FDR of PIP at 1% across all data sets. When analyzing a spike-in data set, PIP-ECHO increases both the accuracy and sensitivity of differential expression analysis, yielding substantially more differentially abundant proteins than either MaxQuant or IonQuant.

Proteomics↗
PubMed2006

Covalent photochemical functionalization of amorphous carbon thin films for integrated real-time biosensing.

Recent studies have demonstrated that carbon, in the form of diamond, can be functionalized with molecular and/or biomolecular species to yield interfaces exhibiting extremely high stability and selectivity in binding to target biomolecules in solution. However, diamond and most other crystalline forms of carbon involve high-temperature deposition or processing steps that restrict their ability to be integrated with other materials. Here, we demonstrate that photochemical functionalization of amorphous carbon films followed by covalent immobilization of DNA yields highly stable surfaces with excellent biomolecular recognition properties that can be used for real-time biological detection. Carbon films deposited onto substrates at 300 K were functionalized with organic alkenes bearing protected amine groups and characterized using X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy. The functionalized carbon surfaces were covalently linked to DNA oligonucleotides. Measurements show very high selectivity for binding to the complementary sequence, and a high density of hybridizing DNA molecules. Samples repeatedly hybridized and denatured 25 times showed no significant degradation. The ability to use amorphous carbon films as a basis for real-time biosensing is demonstrated by coating quartz crystal microbalance (QCM) crystals with a thin carbon film and using this for covalent modification with DNA. Measurements of the resonance frequency show the ability to detect DNA hybridization in real time with a detection limit of <3% of a monolayer, with a high degree of reversibility. These results demonstrate that functionalized films of amorphous carbon can be used as a chemically stable platform for integrated biosensing using only room-temperature processing steps.

Biosensing Techniques↗
PubMed2006

Ionizable isotopic labeling reagent for relative quantification of amine metabolites by mass spectrometry.

A powerful approach to relative quantification by mass spectrometry is to employ labeling reagents that target specific functional groups in molecules of interest. A quantitative comparison of two or more samples may be readily accomplished by using a chemically identical but isotopically distinct labeling reagent for each sample. The samples may then be combined, subjected to purification steps, and mass analyzed. Comparison of the signal intensities obtained from the isotopically labeled variants of the target analyte(s) provides quantitative information on their relative concentrations in the sample. In this report, we describe the synthesis and use of heavy and light isotopic forms of methyl acetimidate for the relative quantification of amine-containing species. The principal advantages of methyl acetimidate as a labeling reagent are that the reaction product is positively charged and hydrophobicity is increased, both of which enhance electrospray ionization efficiency and increase detection sensitivity. The quantitative nature of the analysis was demonstrated in model metabolomics experiments in which heavy and light labeled Arabidopsis extracts were combined in different ratios. Finally, the labeling strategy was employed to determine differences in the amounts of amine-containing metabolites for Arabidopsis seeds germinated under two different conditions.

Amines↗
PubMed2006

Quantitative detection of individual cleaved DNA molecules on surfaces using gold nanoparticles and scanning electron microscope imaging.

Single-nucleotide polymorphisms (SNPs) are the most frequent type of human genetic variation. Recent work has shown that it is possible to directly analyze SNPs in unamplified human genomic DNA samples using the surface-invasive cleavage reaction followed by rolling circle amplification (RCA) labeling of the cleavage products. The individual RCA amplicon molecules were counted on the surface using fluorescence microscopy. Two principal limitations of such single-molecule counting are the variability in the amplicon size, which results in a large variation in fluorescence signal intensity from the dye-labeled DNA molecules, and a high level of background fluorescence. It is shown here that an excellent alternative to RCA labeling is tagging with gold nanoparticles followed by imaging with a scanning electron microscope. Gold nanoparticles have a uniform diameter (15 +/- 0.5 nm) and provide excellent contrast against the background of the silicon substrate employed. Individual gold nanoparticles are readily counted using publicly available software. The results demonstrate that the labeling efficiency is improved by as much as approximately 15-fold, and the signal-to-noise ratio is improved by approximately 4-fold. Detection of individual cleaved DNA molecules following surface-invasive cleavage was linear and quantitative over 3 orders of magnitude in amount of target DNA (10(-18)-10(-15) mol).

DNA↗
PubMed2006

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.

Amino Acid Sequence↗
PubMed2006

Specific capture of mammalian cells by cell surface receptor binding to ligand immobilized on gold thin films.

Aldehyde-terminated self-assembled monolayers (SAMs) on gold surfaces were modified with proteins and employed to capture intact living cells through specific ligand-cell surface receptor interactions. In our model system, the basic fibroblast growth factor (bFGF) binding receptor was targeted on baby hamster kidney (BHK-21) cells. Negative control and target proteins were immobilized on a gold surface by coupling protein primary amines to surface aldehyde groups. Cell-binding was monitored by phase contrast microscopy or surface plasmon resonance (SPR) imaging. The specificity of the receptor-ligand interaction was confirmed by the lack of cell binding to the negative control proteins, cytochrome c and insulin, and by the disruption of cell binding by treatment with heparitinase to destroy heparan sulfate which plays an essential role in the binding of bFGF to FGF receptors. This approach can simultaneously probe a large number of receptor-ligand interactions in cell populations and has potential for targeting and isolating cells from mixtures according to the receptors expressed on their surface.

Animals↗
PubMed2005

Scoring single-nucleotide polymorphisms at the single-molecule level by counting individual DNA cleavage events on surfaces.

Single-nucleotide polymorphisms (SNPs) are the most frequent type of human genetic variation. Recent work has shown that it is possible to directly analyze SNPs in unamplified human genomic DNA samples using the surface-invasive cleavage reaction followed by rolling circle amplification (RCA) of the cleavage products. The ability of RCA to produce single-stranded DNA tens of thousands of nucleotides in length from a single cleaved DNA molecule on the surface suggested the possibility of detecting individual cleavage events on the surface. The feasibility of this approach to SNP scoring is shown here. Individual cleavage events on the surface are detected using fluorescence microscopy to visualize the single-stranded DNA product of the RCA reaction labeled with the fluorescent dye SYBR Green I. The surface density of fluorescent features observed is dependent upon the concentration of target DNA. Future reductions of the sample volume and optimization of the reaction conditions offer the potential of being able to perform such analyses on as little as a single copy of genomic DNA target.

DNA↗
PubMed2005

Controlling gas-phase reactions for efficient charge reduction electrospray mass spectrometry of intact proteins.

Charge reduction electrospray mass spectrometry (CREMS) reduces the charge states of electrospray-generated ions, which concentrates the ions from a protein into fewer peaks spread over a larger m/z range, thereby increasing peak separation and decreasing spectral congestion. An optimized design for a CREMS source is described that provides an order-of-magnitude increase in sensitivity compared to previous designs and provides control over the extent of charge reduction. Either a corona discharge or an alpha-particle source was employed to generate anions that abstract protons from electrosprayed protein cations. These desired ion/ion proton transfer reactions predominated, but some oxidation and ion-attachment reactions also occurred, leading to new peaks or mass-shifted broader peaks while decreasing signal intensity. The species producing these deleterious side-reactions were identified, and conditions were found that prevented their formation. Spectrometer m/z biases were examined because of their effect upon the signal intensity of higher m/z charge-reduced protein ions. The utility of this atmospheric pressure CREMS was demonstrated using a cell lysate fraction from E. coli. The spectral simplification afforded by CREMS reveals more proteins than are observed without charge reduction.

Electrochemistry↗
PubMed2005

Efficient synthesis of a beta-peptide combinatorial library with microwave irradiation.

The predictable relationship between beta-amino acid sequence and folding has inspired several biological applications of beta-peptides. For many such applications, it would be desirable to prepare and screen beta-peptide libraries. However, standard peptide synthesis protocols are not efficient enough to support a library approach for many types of beta-peptides. We recently optimized the solid-phase synthesis of beta-peptides using microwave irradiation, and we have now adapted this approach to synthesis on polystyrene macrobeads. We rapidly prepared a high-quality beta-peptide combinatorial library via a split-and-mix strategy. This library was screened in search of beta-peptide antagonists of the p53-MDM2 protein-protein interaction.

Carbohydrate Sequence↗
PubMed2005

Thermodynamically based DNA strand design.

We describe a new algorithm for design of strand sets, for use in DNA computations or universal microarrays. Our algorithm can design sets that satisfy any of several thermodynamic and combinatorial constraints, which aim to maximize desired hybridizations between strands and their complements, while minimizing undesired cross-hybridizations. To heuristically search for good strand sets, our algorithm uses a conflict-driven stochastic local search approach, which is known to be effective in solving comparable search problems. The PairFold program of Andronescu et al. [M. Andronescu, Z. C. Zhang and A. Condon (2005) J. Mol. Biol., 345, 987-1001; M. Andronescu, R. Aguirre-Hernandez, A. Condon, and H. Hoos (2003) Nucleic Acids Res., 31, 3416-3422.] is used to calculate the minimum free energy of hybridization between two mismatched strands. We describe new thermodynamic measures of the quality of strand sets. With respect to these measures of quality, our algorithm consistently finds, within reasonable time, sets that are significantly better than previously published sets in the literature.

Algorithms↗
PubMed2005

A thermodynamic approach to designing structure-free combinatorial DNA word sets.

An algorithm is presented for the generation of sets of non-interacting DNA sequences, employing existing thermodynamic models for the prediction of duplex stabilities and secondary structures. A DNA 'word' structure is employed in which individual DNA 'words' of a given length (e.g. 12mer and 16mer) may be concatenated into longer sequences (e.g. four tandem words and six tandem words). This approach, where multiple word variants are used at each tandem word position, allows very large sets of non-interacting DNA strands to be assembled from combinations of the individual words. Word sets were generated and their figures of merit are compared to sets as described previously in the literature (e.g. 4, 8, 12, 15 and 16mer). The predicted hybridization behavior was experimentally verified on selected members of the sets using standard UV hyperchromism measurements of duplex melting temperatures (T(m)s). Additional experimental validation was obtained by using the sequences in formulating and solving a small example of a DNA computing problem.

Algorithms↗
PubMed2005

Lectin arrays for profiling cell surface carbohydrate expression.

We present a strategy for the analysis of cell surface carbohydrate expression patterns using lectin arrays fabricated on gold surfaces. Antibody and glycoprotein binding experiments showed that the lectins were effectively immobilized on the surface and retained their carbohydrate-binding specificities. The approach was demonstrated in the analysis of carbohydrate expression on two mammalian cell lines.

Animals↗
PubMed2005

Tripeptidyl peptidase II. An oligomeric protease complex from Arabidopsis.

The breakdown of most nuclear and cytoplasmic proteins involves their partial cleavage by the 26S proteasome followed by further disassembly to free amino acids by the combined action of endo- and exopeptidases. In animals, one important intermediate exopeptidase is tripeptidyl peptidase (TPP)II, which digests peptide products of the 26S proteasome and other endopeptidases into tripeptides. Here, we describe the purification and characterization of TPPII from Arabidopsis (Arabidopsis thaliana). Like its animal counterparts, Arabidopsis TPPII exists as a soluble, approximately 5- to 9-MD complex. Two related species of 153 and 142 kD are present in the purified preparations that are derived from a single TPP2 gene. Sequencing by Edman degradation of the intact polypeptides and mass spectrometry of proteolytic fragments demonstrated that the 142-kD form mainly differs from the 153-kD form by a truncation at the C-terminal end. This serine protease is a member of the subtilisin superfamily and is sensitive to the inhibitors alanine-alanine-phenylalanine-chloromethylketone and butabindide, which are diagnostic for the TPPII subfamily. The Arabidopsis TPP2 gene is widely expressed in many tissue types with related genes evident in other plant genomes. Whereas the 26S proteasome is essential, TPPII appears not as important for plant physiology. An Arabidopsis T-DNA mutant defective in TPP2 expression displays no phenotypic abnormalities and is not hypersensitive to either amino acid analogs or the 26S proteasome inhibitor MG132. As a consequence, plants likely contain other intermediate exopeptidases that assist in amino acid recycling.

Amino Acid Sequence↗
PubMed2005

Parallel single nucleotide polymorphism genotyping by surface invasive cleavage with universal detection.

Large-scale investigations of sequence variation within the human species will provide information about the basis of heritable variation in disease susceptibility and human migration. The surface invader assay (an adaptation of the invasive cleavage reaction to an array format) is capable of exquisitely sensitive and specific detection of genetic variation. It is shown here that this genotyping technology can be multiplexed in a DNA array format, permitting the parallel analysis of a panel of single nucleotide polymorphisms (SNPs) directly from an unamplified genomic DNA target. In addition, a "universal" mode of detection was developed that makes use of a mixture of degenerate templates for DNA ligation to the surface-bound cleaved oligonucleotides and thereby makes this strategy amenable to any desired SNP site or combination of SNP sites, without regard to their particular DNA sequences. This approach was demonstrated on a proof-of-principle scale using small DNA arrays to genotype 6 SNP markers in the PTPN1 gene and 10 mutations in the cystic fibrosis transmembrane conductance regulator gene. This ability to analyze many different genetic variations in parallel, directly from unamplified human genomic DNA samples, lays the groundwork for the development of high-density arrays able to analyze hundreds of thousands or even millions of SNPs.

Base Sequence↗
PubMed2005

Alpha-Ketoisocaproate-induced hypersecretion of insulin by islets from diabetes-susceptible mice.

Most patients at risk for developing type 2 diabetes are hyperinsulinemic. Hyperinsulinemia may be a response to insulin resistance, but another possible abnormality is insulin hypersecretion. BTBR mice are insulin resistant and hyperinsulinemic. When the leptin(ob) mutation is introgressed into BTBR mice, they develop severe diabetes. We compared the responsiveness of lean B6 and BTBR mouse islets to various insulin secretagogues. The transamination product of leucine, alpha-ketoisocaproate (KIC), elicited a dramatic insulin secretory response in BTBR islets. The KIC response was blocked by methyl-leucine or aminooxyacetate, inhibitors of branched-chain amino transferase. When dimethylglutamate was combined with KIC, the fractional insulin secretion was identical in islets from both mouse strains, predicting that the amine donor is rate-limiting for KIC-induced insulin secretion. Consistent with this prediction, glutamate levels were higher in BTBR than in B6 islets. The transamination product of glutamate, alpha-ketoglutarate, elicited insulin secretion equally from B6 and BTBR islets. Thus formation of alpha-ketoglutarate is a requisite step in the response of mouse islets to KIC. alpha-Ketoglutarate can be oxidized to succinate. However, succinate does not stimulate insulin secretion in mouse islets. Our data suggest that alpha-ketoglutarate may directly stimulate insulin secretion and that increased formation of alpha-ketoglutarate leads to hyperinsulinemia.

Animals↗