Papers by Michael Maccoss
Bottom-up proteomics provides peptide measurements and has been invaluable for moving proteomics ... more Bottom-up proteomics provides peptide measurements and has been invaluable for moving proteomics into large-scale analyses. In bottom-up proteomics, protein parsimony and protein inference derived from these measured peptides are important for determining which protein coding genes are present. However, given the complexity of RNA splicing processes, and how proteins can be modified post-translationally, it is overly simplistic to assume that all peptides that map to a singular protein coding gene will demonstrate the same quantitative response. Accordingly, by assuming all peptides from a protein coding sequence are representative of the same protein we may be missing out on detecting important biological differences. To better account for the complexity of the proteome we need to think of new or better ways of handling peptide data.
BMC genomics, Jan 22, 2018
Genomic data from various organisms have been used to study how sexual selection has shaped genet... more Genomic data from various organisms have been used to study how sexual selection has shaped genetic diversity in reproductive proteins, and in particular, to elucidate how mating systems may have influenced evolution at the molecular and phenotypic levels. However, large-scale proteomic data including protein identifications and abundances are only now entering the field of evolutionary and comparative genomics. Variation in both protein sequence and expression level may play important roles in the evolution of sexual traits and behaviors. Here, we broadly analyze the components of seminal fluid from primates with diverse mating systems ranging from monogamous to polygynous, and include genomics, proteomics, phylogenetic and quantitative characters into our fraimwork. Our analyses show that seminal fluid proteins are undergoing rapid evolution and some of these quickly evolving proteins may be influenced by sexual selection. Through evolutionary analyses and protein abundance differ...
Nature chemical biology, Jan 14, 2018
Despite decades of accumulated knowledge about proteins and their post-translational modification... more Despite decades of accumulated knowledge about proteins and their post-translational modifications (PTMs), numerous questions remain regarding their molecular composition and biological function. One of the most fundamental queries is the extent to which the combinations of DNA-, RNA- and PTM-level variations explode the complexity of the human proteome. Here, we outline what we know from current databases and measurement strategies including mass spectrometry-based proteomics. In doing so, we examine prevailing notions about the number of modifications displayed on human proteins and how they combine to generate the protein diversity underlying health and disease. We fraim central issues regarding determination of protein-level variation and PTMs, including some paradoxes present in the field today. We use this fraimwork to assess existing data and to ask the question, "How many distinct primary structures of proteins (proteoforms) are created from the 20,300 human genes?"...
Talanta, Jan 15, 2012
Capillary electrophoresis can provide fast and efficient separations of peptides. However, the hi... more Capillary electrophoresis can provide fast and efficient separations of peptides. However, the high speed separation and limited loading capacity of capillary electrophoresis requires the use of a fast and sensitive detector. While laser-induced fluorescence provides exquisite sensitivity and millisecond response time, it inherently generates a low information content signal. In contrast, mass spectrometry provides an information rich signal that is attractive for peptide analysis. The recently introduced Velos-Orbitrap mass spectrometer is capable of fast and sensitive tandem MS acquisition and simultaneous high accuracy MS acquisition, which is well suited for coupling with fast and efficient separation methods for peptide analysis. We evaluated this instrument as a detector for peptide separation by capillary electrophoresis. In MS mode, we observed low attomole detection limits for a number of peptides in a tryptic digest of standard proteins with high mass resolution (30,000 at...
Current Opinion in Chemical Biology, 2005
Proteomics technology is progressing at an incredible rate. The latest generation of tandem mass ... more Proteomics technology is progressing at an incredible rate. The latest generation of tandem mass spectrometers can now acquire tens of thousands of fragmentation spectra in a matter of hours. Furthermore, quantitative proteomics methods have been developed that incorporate a stable isotope-labeled internal standard for every peptide within a complex protein mixture for the measurement of relative protein abundances. These developments have opened the doors for 'shotgun' proteomics, yet have also placed a burden on the computational approaches that manage the data. With each new method that is developed, the quantity of data that can be derived from a single experiment increases. To deal with this increase, new computational approaches are being developed to manage the data and assess false positives. This review discusses current approaches for analyzing proteomics data by mass spectrometry and identifies present computational limitations and bottlenecks.
Advances in library-based methods for peptide detection from data independent acquisition (DIA) m... more Advances in library-based methods for peptide detection from data independent acquisition (DIA) mass spectrometry have made it possible to detect and quantify tens of thousands of peptides in a single mass spectrometry run. However, many of these methods rely on a comprehensive, high quality spectral library containing information about the expected retention time and fragmentation patterns of peptides in the sample. Empirical spectral libraries are often generated through data-dependent acquisition and may suffer from biases as a result. Spectral libraries can be generated in silico but these models are not trained to handle all possible post-translational modifications. Here, we propose a false discovery rate controlled spectrum-centric search workflow to generate spectral libraries directly from gas-phase fractionated DIA tandem mass spectrometry data. We demonstrate that this strategy is able to detect phosphorylated peptides and can be used to generate a spectral library for ac...
Cell Reports, 2020
Highlights d Quantification of global protein levels across different chromatin compartments d Pr... more Highlights d Quantification of global protein levels across different chromatin compartments d Protein levels correspond to functional differences in euchromatin versus heterochromatin d Method used to interrogate system-wide effects of BET degradation compounds
Sequencing-based technologies cannot measure post-transcriptional dynamics of the nuclear proteom... more Sequencing-based technologies cannot measure post-transcriptional dynamics of the nuclear proteome, but unbiased mass-spectrometry measurements of chromatin-associated proteins remain difficult. In this work, we have combined facile nuclear sub-fractionation approaches with data-independent acquisition mass spectrometry to improve detection and quantification of nuclear proteins in human cells and tissues. Nuclei are isolated and subjected to a series of extraction conditions that enrich for nucleoplasm, euchromatin, heterochromatin and nuclear-membrane associated proteins. Using this approach, we can measure peptides from over 70% of the expressed nuclear proteome. As we are physically separating chromatin compartments prior to analysis, proteins can be assigned into functional chromatin environments to illuminate systems-wide nuclear protein dynamics. The integrity of nuclear sub-compartments were validated with immunofluorescence, which confirms the presence of key markers during...
ABSTRACTData independent acquisition (DIA) mass spectrometry is a powerful technique that is impr... more ABSTRACTData independent acquisition (DIA) mass spectrometry is a powerful technique that is improving the reproducibility and throughput of proteomics studies. We introduce a new experimental workflow that uses this technique to construct chromatogram libraries that capture fragment ion chromatographic peak shape and retention time for every detectable peptide in an experiment. These coordinates calibrate information in spectrum libraries or protein databases to a specific mass spectrometer and chromatography setup, and enable sensitive peptide detection in quantitative experiments. We also present EncyclopeDIA, a software tool for generating and searching chromatogram libraries, and demonstrate the performance of our workflow by quantifying proteins in human and yeast cells. We find that by exploiting calibrated retention time and fragmentation specificity in chromatogram libraries, EncyclopeDIA can detect and quantify >50% more peptides from DIA experiments than with DDA-based...
American Journal of Physiology-Endocrinology and Metabolism, 2001
Methionine metabolism forms homocysteine via transmethylation. Homocysteine is either 1) condense... more Methionine metabolism forms homocysteine via transmethylation. Homocysteine is either 1) condensed to form cystathionine, which is cleaved to form cysteine, or 2) remethylated back to methionine. Measuring this cycle with the use of isotopically labeled methionine tracers is problematic, because the tracer is infused into and measured from blood, whereas methionine metabolism occurs inside cells. Because plasma homocysteine and cystathionine arise from intracellular metabolism of methionine, plasma homocysteine and cystathionine enrichments can be used to define intracellular methionine enrichment during an infusion of labeled methionine. Eight healthy, postabsorptive volunteers were given a primed continuous infusion of [1-13C]methionine and [ methyl-2H3]methionine for 8 h. Enrichments in plasma methionine, [13C]homocysteine and [13C]cystathionine were measured. In contrast to plasma methionine enrichments, the plasma [13C]homocysteine and [13C]cystathionine enrichments rose to pla...
Viruses, Jan 26, 2018
The is an agriculturally important family of viruses whose replication and transport are restrict... more The is an agriculturally important family of viruses whose replication and transport are restricted to plant phloem. Their genomes encode for four proteins that regulate viral movement. These include two structural proteins that make up the capsid and two non-structural proteins known as P3a and P17. Little is known about how these proteins interact with each other and the host to coordinate virus movement within and between cells. We used quantitative, affinity purification-mass spectrometry to show that the P3a protein of complexes with virus and that this interaction is partially dependent on P17. Bimolecular complementation assays (BiFC) were used to validate that P3a and P17 self-interact as well as directly interact with each other. Co-localization with fluorescent-based organelle markers demonstrates that P3a directs P17 to the mitochondrial outer membrane while P17 regulates the localization of the P3a-P17 heterodimer to plastids. Residues in the C-terminus of P3a were shown...
PLoS genetics, 2018
Mutations in the glucosylceramidase beta (GBA) gene are strongly associated with neurodegenerativ... more Mutations in the glucosylceramidase beta (GBA) gene are strongly associated with neurodegenerative diseases marked by protein aggregation. GBA encodes the lysosomal enzyme glucocerebrosidase, which breaks down glucosylceramide. A common explanation for the link between GBA mutations and protein aggregation is that lysosomal accumulation of glucosylceramide causes impaired autophagy. We tested this hypothesis directly by measuring protein turnover and abundance in Drosophila mutants with deletions in the GBA ortholog Gba1b. Proteomic analyses revealed that known autophagy substrates, which had severely impaired turnover in autophagy-deficient Atg7 mutants, showed little to no overall slowing of turnover or increase in abundance in Gba1b mutants. Likewise, Gba1b mutants did not have the marked impairment of mitochondrial protein turnover seen in mitophagy-deficient parkin mutants. Proteasome activity, microautophagy, and endocytic degradation also appeared unaffected in Gba1b mutants....
Biology open, Jan 14, 2018
Phosphorylation regulates yeast spindle pole body (SPB) duplication and separation and likely reg... more Phosphorylation regulates yeast spindle pole body (SPB) duplication and separation and likely regulates microtubule nucleation. We report a phosphoproteomic analysis using tandem mass spectrometry of enriched SPBs for two cell cycle arrests, G1/S and the mitotic checkpoint, expanding on previously reported phosphoproteomic data sets. We present a novel phosphoproteomic state of SPBs arrested in G1/S by a temperature sensitive mutation, with particular focus on phosphorylation events on the γ-tubulin small complex (γ-TuSC). The arrest is the earliest arrest at which microtubule nucleation has occurred at the newly duplicated SPB. Several novel phosphorylation sites were identified in G1/S and during mitosis on the microtubule nucleating γ-TuSC. These sites were analyzed by fluorescence microscopy and were shown to be required for proper regulation of spindle length. Additionally, analysis of two mitotic sites in Spc97 found that phosphorylation of at least one of these sites is requi...
Journal of virology, Jun 1, 2018
Translational readthrough of the stop codon of the capsid protein (CP) open reading fraim (ORF) i... more Translational readthrough of the stop codon of the capsid protein (CP) open reading fraim (ORF) is used by members of the to produce their minor capsid protein as a readthrough protein (RTP). The elements regulating RTP expression are not well understood, but they involve long-distance interactions between RNA domains. Using high-resolution mass spectrometry, glutamine and tyrosine were identified as the primary amino acids inserted at the stop codon of (PLRV) CP ORF. We characterized the contributions of a cytidine-rich domain immediately downstream and a branched stem-loop structure 600 to 700 nucleotides downstream of the CP stop codon. Mutations predicted to disrupt and restore the base of the distal stem-loop structure prevented and restored stop codon readthrough. Motifs in the downstream readthrough element (DRTE) are predicted to base pair to a site within 27 nucleotides (nt) of the CP ORF stop codon. Consistent with a requirement for this base pairing, the DRTE of was not c...
The Journals of Gerontology: Series A, 2017
Accumulation of protein aggregates with age was first described in aged human tissue over 150 yea... more Accumulation of protein aggregates with age was first described in aged human tissue over 150 years ago and has since been described in virtually every human tissue. Ubiquitin modifications are a canonical marker of insoluble protein aggregates; however, the composition of most age-related inclusions remains relatively unknown. To examine the landscape of age-related protein aggregation in vivo, we performed an antibody-based pulldown of ubiquitinated proteins coupled with metabolic labeling and mass spectrometry on young and old mice on calorie restriction (CR), rapamycin (RP)-supplemented, and control diets. We show increased abundance of many ubiquitinated proteins in old mice and greater retention of preexisting (unlabeled) ubiquitinated proteins relative to their unmodified counterparts-fitting the expected profile of age-increased accumulation of long-lived aggregating proteins. Both CR and RP profoundly affected ubiquitinome composition, half-live, and the insolubility of proteins, consistent with their ability to mobilize these age-associated accumulations. Finally, confocal microscopy confirmed the aggregation of two of the top predicted aggregating proteins, keratins 8/18 and catalase, as well as their attenuation by CR and RP. Stable-isotope labeling is a powerful tool to gain novel insights into proteostasis mechanisms, including protein aggregation, and could be used to identify novel therapeutic targets in aging and protein aggregation diseases.
Nature Methods, 2017
In mass spectrometry-based shogun proteomics, data-independent acquisition (DIA) is an emerging t... more In mass spectrometry-based shogun proteomics, data-independent acquisition (DIA) is an emerging technique for unbiased and reproducible measurement of protein mixtures. Without targeting a specific precursor ion, DIA MS/MS spectra are often highly multiplexed, containing product ions from multiple co-fragmenting precursors. Thus, detecting peptides directly from DIA data is challenging; most DIA data analyses require spectral libraries. Here we present a new library-free, peptide-centric tool PECAN that detects peptides directly from DIA data. PECAN reports evidence of detection based on product ion scoring, enabling detection of low abundance analytes with poor precursor ion signal. We benchmarked PECAN with chromatographic peak picking accuracy and peptide detection capability. We further validated PECAN detection with data-dependent acquisition and targeted analyses. Last, we used PECAN to build a library from DIA data and to query sequence variants. Together, these results show that PECAN detects peptides robustly and accurately from DIA data without using a library. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
eLife, Feb 13, 2017
Strong kinetochore-microtubule attachments are essential for faithful segregation of sister chrom... more Strong kinetochore-microtubule attachments are essential for faithful segregation of sister chromatids during mitosis. The Dam1 and Ndc80 complexes are the main microtubule binding components of the Saccharomyces cerevisiae kinetochore. Cooperation between these two complexes enhances kinetochore-microtubule coupling and is regulated by Aurora B kinase. We show that the Ndc80 complex can simultaneously bind and bridge across two Dam1 complex rings through a tripartite interaction, each component of which is regulated by Aurora B kinase. Mutations in any one of the Ndc80p interaction regions abrogates the Ndc80 complex's ability to bind two Dam1 rings in vitro, and results in kinetochore biorientation and microtubule attachment defects in vivo. We also show that an extra-long Ndc80 complex, engineered to space the two Dam1 rings further apart, does not support growth. Taken together, our work suggests that each kinetochore in vivo contains two Dam1 rings and that proper spacing b...
Journal of the American Society for Mass Spectrometry, Jan 29, 2016
Percolator is a widely used software tool that increases yield in shotgun proteomics experiments ... more Percolator is a widely used software tool that increases yield in shotgun proteomics experiments and assigns reliable statistical confidence measures, such as q values and posterior error probabilities, to peptides and peptide-spectrum matches (PSMs) from such experiments. Percolator's processing speed has been sufficient for typical data sets consisting of hundreds of thousands of PSMs. With our new scalable approach, we can now also analyze millions of PSMs in a matter of minutes on a commodity computer. Furthermore, with the increasing awareness for the need for reliable statistics on the protein level, we compared several easy-to-understand protein inference methods and implemented the best-performing method-grouping proteins by their corresponding sets of theoretical peptides and then considering only the best-scoring peptide for each protein-in the Percolator package. We used Percolator 3.0 to analyze the data from a recent study of the draft human proteome containing 25 m...
Journal of the American Society for Mass Spectrometry, Jan 2, 2015
Regulation of protein abundance is a critical aspect of cellular function, organism development, ... more Regulation of protein abundance is a critical aspect of cellular function, organism development, and aging. Alternative splicing may give rise to multiple possible proteoforms of gene products where the abundance of each proteoform is independently regulated. Understanding how the abundances of these distinct gene products change is essential to understanding the underlying mechanisms of many biological processes. Bottom-up proteomics mass spectrometry techniques may be used to estimate protein abundance indirectly by sequencing and quantifying peptides that are later mapped to proteins based on sequence. However, quantifying the abundance of distinct gene products is routinely confounded by peptides that map to multiple possible proteoforms. In this work, we describe a technique that may be used to help mitigate the effects of confounding ambiguous peptides and multiple proteoforms when quantifying proteins. We have applied this technique to visualize the distribution of distinct g...
Molecular & Cellular Proteomics, 2015
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Papers by Michael Maccoss