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Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

St. Jude Children's Research Hospital

Protein ubiquitination is an essential post-translational modification regulating neurodevelopment, synaptic plasticity, learning, and memory, and its dysregulation contributes to the pathogenesis of neurological diseases. Here we report a systematic analysis of ubiquitinated proteome (ubiquitome) in rat brain using a newly developed monoclonal antibody that recognizes the diglycine tag on lysine residues in trypsinized peptides (K-GG peptides). Initial antibody specificity analysis showed that the antibody can distinguish K-GG peptides from linear GG peptides or pseudo K-GG peptides derived from iodoacetamide. To evaluate the false discovery rate of K-GG peptide matches during database search, we introduced a null experiment using bacterial lysate that contains no such peptides. The brain ubiquitome was then analyzed by this antibody enrichment with or without strong cation exchange (SCX) prefractionation. During SCX chromatography, although the vast majority of K-GG peptides were detected in the fractions containing at least three positive charged peptides, specific K-GG peptides with two positive charges (e.g., protein N-terminal acetylated and C-terminal non-K/R peptides) were also identified in early fractions. The reliability of C-terminal K-GG peptides was also extensively investigated. Finally, we collected a data set of 1786 K-GG sites on 2064 peptides in 921 proteins and estimated their abundance by spectral counting. The study reveals a wide range of ubiquitination events on key components in presynaptic region (e.g., Bassoon, NSF, SNAP25, synapsin, synaptotagmin, and syntaxin) and postsynaptic density (e.g., PSD-95, GKAP, CaMKII, as well as receptors for NMDA, AMPA, GABA, serotonin, and acetylcholine). We also determined ubiquitination sites on amyloid precursor protein and alpha synuclein that are thought to be causative agents in Alzhermer's and Parkinson's disorders, respectively. As K-GG peptides can also be produced from Nedd8 or ISG15 modified proteins, we quantified these proteins in the brain and found that their levels are less than 2% of ubiquitin. Together, this study demonstrates that a large number of neuronal proteins are modified by ubiquitination and provides a feasible method for profiling the ubiquitome in the brain.

Synaptic protein ubiquitination in rat brain revealed by antibody-based ubiquitome analysis.

Deposition of insoluble protein aggregates is a hallmark of neurodegenerative diseases. The universal presence of β-amyloid and tau in Alzheimer's disease (AD) has facilitated advancement of the amyloid cascade and tau hypotheses that have dominated AD pathogenesis research and therapeutic development. However, the underlying etiology of the disease remains to be fully elucidated. Here we report a comprehensive study of the human brain-insoluble proteome in AD by mass spectrometry. We identify 4,216 proteins, among which 36 proteins accumulate in the disease, including U1-70K and other U1 small nuclear ribonucleoprotein (U1 snRNP) spliceosome components. Similar accumulations in mild cognitive impairment cases indicate that spliceosome changes occur in early stages of AD. Multiple U1 snRNP subunits form cytoplasmic tangle-like structures in AD but not in other examined neurodegenerative disorders, including Parkinson disease and frontotemporal lobar degeneration. Comparison of RNA from AD and control brains reveals dysregulated RNA processing with accumulation of unspliced RNA species in AD, including myc box-dependent-interacting protein 1, clusterin, and presenilin-1. U1-70K knockdown or antisense oligonucleotide inhibition of U1 snRNP increases the protein level of amyloid precursor protein. Thus, our results demonstrate unique U1 snRNP pathology and implicate abnormal RNA splicing in AD pathogenesis.

U1 small nuclear ribonucleoprotein complex and RNA splicing alterations in Alzheimer's disease.

What proportion of genes with intense and selective expression in specific tissues, cells, or systems are still almost completely uncharacterized with respect to biological function? In what ways do these functionally enigmatic genes differ from well-studied genes? To address these two questions, we devised a computational approach that defines so-called ignoromes. As proof of principle, we extracted and analyzed a large subset of genes with intense and selective expression in brain. We find that publications associated with this set are highly skewed--the top 5% of genes absorb 70% of the relevant literature. In contrast, approximately 20% of genes have essentially no neuroscience literature. Analysis of the ignorome over the past decade demonstrates that it is stubbornly persistent, and the rapid expansion of the neuroscience literature has not had the expected effect on numbers of these genes. Surprisingly, ignorome genes do not differ from well-studied genes in terms of connectivity in coexpression networks. Nor do they differ with respect to numbers of orthologs, paralogs, or protein domains. The major distinguishing characteristic between these sets of genes is date of discovery, early discovery being associated with greater research momentum--a genomic bandwagon effect. Finally we ask to what extent massive genomic, imaging, and phenotype data sets can be used to provide high-throughput functional annotation for an entire ignorome. In a majority of cases we have been able to extract and add significant information for these neglected genes. In several cases--ELMOD1, TMEM88B, and DZANK1--we have exploited sequence polymorphisms, large phenome data sets, and reverse genetic methods to evaluate the function of ignorome genes.

Functionally enigmatic genes: a case study of the brain ignorome.

Database search programs are essential tools for identifying peptides via mass spectrometry (MS) in shotgun proteomics. Simultaneously achieving high sensitivity and high specificity during a database search is crucial for improving proteome coverage. Here we present JUMP, a new hybrid database search program that generates amino acid tags and ranks peptide spectrum matches (PSMs) by an integrated score from the tags and pattern matching. In a typical run of liquid chromatography coupled with high-resolution tandem MS, more than 95% of MS/MS spectra can generate at least one tag, whereas the remaining spectra are usually too poor to derive genuine PSMs. To enhance search sensitivity, the JUMP program enables the use of tags as short as one amino acid. Using a target-decoy strategy, we compared JUMP with other programs (e.g. SEQUEST, Mascot, PEAKS DB, and InsPecT) in the analysis of multiple datasets and found that JUMP outperformed these preexisting programs. JUMP also permitted the analysis of multiple co-fragmented peptides from "mixture spectra" to further increase PSMs. In addition, JUMP-derived tags allowed partial de novo sequencing and facilitated the unambiguous assignment of modified residues. In summary, JUMP is an effective database search algorithm complementary to current search programs.

JUMP: a tag-based database search tool for peptide identification with high sensitivity and accuracy.

Alzheimer's disease (AD) is the most common form of dementia, characterized by progressive loss of cognitive function. One of the pathological hallmarks of AD is the formation of neurofibrillary tangles composed of abnormally hyperphosphorylated tau protein, but global deregulation of protein phosphorylation in AD is not well analyzed. Here, we report a pilot investigation of AD phosphoproteome by titanium dioxide enrichment coupled with high resolution LC-MS/MS. During the optimization of the enrichment method, we found that phosphate ion at a low concentration (e.g. 1 mM) worked efficiently as a nonphosphopeptide competitor to reduce background. The procedure was further tuned with respect to peptide-to-bead ratio, phosphopeptide recovery, and purity. Using this refined method and 9 h LC-MS/MS, we analyzed phosphoproteome in one milligram of digested AD brain lysate, identifying 5243 phosphopeptides containing 3715 nonredundant phosphosites on 1455 proteins, including 31 phosphosites on the tau protein. This modified enrichment method is simple and highly efficient. The AD case study demonstrates its feasibility of dissecting phosphoproteome in a limited amount of postmortem human brain. All MS data have been deposited in the ProteomeXchange with identifier PXD001180 (http://proteomecentral.proteomexchange.org/dataset/PXD001180).

Refined phosphopeptide enrichment by phosphate additive and the analysis of human brain phosphoproteome.

The development of high-resolution liquid chromatography (LC) is essential for improving the sensitivity and throughput of mass spectrometry (MS)-based proteomics. Here we present systematic optimization of a long gradient LC-MS/MS platform to enhance protein identification from a complex mixture. The platform employed an in-house fabricated, reverse-phase long column (100 μm × 150 cm, 5 μm C18 beads) coupled to Q Exactive MS. The column was capable of achieving a peak capacity of ∼700 in a 720 min gradient of 10-45% acetonitrile. The optimal loading level was ∼6 μg of peptides, although the column allowed loading as many as 20 μg. Gas-phase fractionation of peptide ions further increased the number of peptide identification by ∼10%. Moreover, the combination of basic pH LC prefractionation with the long gradient LC-MS/MS platform enabled the identification of 96,127 peptides and 10,544 proteins at 1% protein false discovery rate in a post-mortem brain sample of Alzheimer's disease. Because deep RNA sequencing of the same specimen suggested that ∼16,000 genes were expressed, the current analysis covered more than 60% of the expressed proteome. Further improvement strategies of the LC/LC-MS/MS platform were also discussed.

Systematic optimization of long gradient chromatography mass spectrometry for deep analysis of brain proteome.

Chemotactic migration of fibroblasts toward growth factors relies on their capacity to sense minute extracellular gradients and respond to spatially confined receptor-mediated signals. Currently, mechanisms underlying the gradient sensing of fibroblasts remain poorly understood. Using single-particle tracking methodology, we determined that a lysophosphatidic acid (LPA) gradient induces a spatiotemporally restricted decrease in the mobility of LPA receptor 2 (LPA2) on chemotactic fibroblasts. The onset of decreased LPA2 mobility correlates to the spatial recruitment and coupling to LPA2-interacting proteins that anchor the complex to the cytoskeleton. These localized PDZ motif-mediated macromolecular complexes of LPA2 trigger a Ca(2+) puff gradient that governs gradient sensing and directional migration in response to LPA. Disruption of the PDZ motif-mediated assembly of the macromolecular complex of LPA2 disorganizes the gradient of Ca(2+) puffs, disrupts gradient sensing, and reduces the directional migration of fibroblasts toward LPA. Our findings illustrate that the asymmetric macromolecular complex formation of chemoattractant receptors mediates gradient sensing and provides a new mechanistic basis for models to describe gradient sensing of fibroblasts.

Asymmetrical macromolecular complex formation of lysophosphatidic acid receptor 2 (LPA2) mediates gradient sensing in fibroblasts.

Mass spectrometry is one of the most sensitive methods in analytical chemistry, and its application in proteomics has been rapidly expanded after sequencing the human genome. Mass spectrometry is now the mainstream approach for identification and quantification of proteins and posttranslational modifications, either in small scale or in the entire proteome. Shotgun proteomics can analyze up to 10,000 proteins in a comprehensive study, with detection sensitivity in the picogram range. In this chapter, we describe major experimental steps in a shotgun proteomics platform, including sample preparation in the context of studying protein-protein interaction, mass spectrometric data acquisition, and database search to identify proteins and posttranslational modification analysis. Proteome quantification strategies and bioinformatics analysis are also illustrated. Finally, we discuss the capabilities, limitations, and potential improvements of current platforms.

Quantitative protein analysis by mass spectrometry.

The mind bomb 1 (Mib1) ubiquitin ligase is essential for controlling metazoan development by Notch signaling and possibly the Wnt pathway. It is also expressed in postmitotic neurons and regulates neuronal morphogenesis and synaptic activity by mechanisms that are largely unknown. We sought to comprehensively characterize the Mib1 interactome and study its potential function in neuron development utilizing a novel sequential elution strategy for affinity purification, in which Mib1 binding proteins were eluted under different stringency and then quantified by the isobaric labeling method. The strategy identified the Mib1 interactome with both deep coverage and the ability to distinguish high-affinity partners from low-affinity partners. A total of 817 proteins were identified during the Mib1 affinity purification, including 56 high-affinity partners and 335 low-affinity partners, whereas the remaining 426 proteins are likely copurified contaminants or extremely weak binding proteins. The analysis detected all previously known Mib1-interacting proteins and revealed a large number of novel components involved in Notch and Wnt pathways, endocytosis and vesicle transport, the ubiquitin-proteasome system, cellular morphogenesis, and synaptic activities. Immunofluorescence studies further showed colocalization of Mib1 with five selected proteins: the Usp9x (FAM) deubiquitinating enzyme, alpha-, beta-, and delta-catenins, and CDKL5. Mutations of CDKL5 are associated with early infantile epileptic encephalopathy-2 (EIEE2), a severe form of mental retardation. We found that the expression of Mib1 down-regulated the protein level of CDKL5 by ubiquitination, and antagonized CDKL5 function during the formation of dendritic spines. Thus, the sequential elution strategy enables biochemical characterization of protein interactomes; and Mib1 analysis provides a comprehensive interactome for investigating its role in signaling networks and neuronal development.

Sequential Elution Interactome Analysis of the Mind Bomb 1 Ubiquitin Ligase Reveals a Novel Role in Dendritic Spine Outgrowth.

Ulcerative colitis (UC) belongs to inflammatory bowel disorders, a group of gastrointestinal disorders that can produce serious recurring diarrhea in affected patients. The mechanism for UC- and inflammatory bowel disorder-associated diarrhea is not well understood. The cystic fibrosis transmembrane-conductance regulator (CFTR) chloride channel plays an important role in fluid and water transport across the intestinal mucosa. CFTR channel function is regulated in a compartmentalized manner through the formation of CFTR-containing macromolecular complexes at the plasma membrane. In this study, we demonstrate the involvement of a novel macromolecular signaling pathway that causes diarrhea in UC. We found that a nitric oxide-producing enzyme, inducible nitric oxide synthase (iNOS), is overexpressed under the plasma membrane and generates compartmentalized cGMP in gut epithelia in UC. The scaffolding protein Na(+)/H(+) exchanger regulatory factor 2 (NHERF2) bridges iNOS with CFTR, forming CFTR-NHERF2-iNOS macromolecular complexes that potentiate CFTR channel function via the nitric oxide-cGMP pathway under inflammatory conditions both in vitro and in vivo. Potential disruption of these complexes in Nherf2(-/-) mice may render them more resistant to CFTR-mediated secretory diarrhea than Nherf2(+/+) mice in murine colitis models. Our study provides insight into the mechanism of pathophysiologic occurrence of diarrhea in UC and suggests that targeting CFTR and CFTR-containing macromolecular complexes will ameliorate diarrheal symptoms and improve conditions associated with inflammatory bowel disorders.

Altered cGMP dynamics at the plasma membrane contribute to diarrhea in ulcerative colitis.

Phenome-wide association is a novel reverse genetic strategy to analyze genome-to-phenome relations in human clinical cohorts. Here we test this approach using a large murine population segregating for ∼5 million sequence variants, and we compare our results to those extracted from a matched analysis of gene variants in a large human cohort. For the mouse cohort, we amassed a deep and broad open-access phenome consisting of ∼4,500 metabolic, physiological, pharmacological and behavioural traits, and more than 90 independent expression quantitative trait locus (QTL), transcriptome, proteome, metagenome and metabolome data sets--by far the largest coherent phenome for any experimental cohort (www.genenetwork.org). We tested downstream effects of subsets of variants and discovered several novel associations, including a missense mutation in fumarate hydratase that controls variation in the mitochondrial unfolded protein response in both mouse and Caenorhabditis elegans, and missense mutations in Col6a5 that underlies variation in bone mineral density in both mouse and human.

Joint mouse-human phenome-wide association to test gene function and disease risk.

Proteogenomics is an emerging approach to improve gene annotation and interpretation of proteomics data. Here we present JUMPg, an integrative proteogenomics pipeline including customized database construction, tag-based database search, peptide-spectrum match filtering, and data visualization. JUMPg creates multiple databases of DNA polymorphisms, mutations, splice junctions, partially trypticity, as well as protein fragments translated from the whole transcriptome in all six frames upon RNA-seq de novo assembly. We use a multistage strategy to search these databases sequentially, in which the performance is optimized by re-searching only unmatched high-quality spectra and reusing amino acid tags generated by the JUMP search engine. The identified peptides/proteins are displayed with gene loci using the UCSC genome browser. Then, the JUMPg program is applied to process a label-free mass spectrometry data set of Alzheimer's disease postmortem brain, uncovering 496 new peptides of amino acid substitutions, alternative splicing, frame shift, and "non-coding gene" translation. The novel protein PNMA6BL specifically expressed in the brain is highlighted. We also tested JUMPg to analyze a stable-isotope labeled data set of multiple myeloma cells, revealing 991 sample-specific peptides that include protein sequences in the immunoglobulin light chain variable region. Thus, the JUMPg program is an effective proteogenomics tool for multiomics data integration.

JUMPg: An Integrative Proteogenomics Pipeline Identifying Unannotated Proteins in Human Brain and Cancer Cells.

Isobaric labeling quantification by mass spectrometry (MS) has emerged as a powerful technology for multiplexed large-scale protein profiling, but measurement accuracy in complex mixtures is confounded by the interference from coisolated ions, resulting in ratio compression. Here we report that the ratio compression can be essentially resolved by the combination of pre-MS peptide fractionation, MS2-based interference detection, and post-MS computational interference correction. To recapitulate the complexity of biological samples, we pooled tandem mass tag (TMT)-labeled Escherichia coli peptides at 1:3:10 ratios and added in ∼20-fold more rat peptides as background, followed by the analysis of two-dimensional liquid chromatography (LC)-MS/MS. Systematic investigation shows that quantitative interference was impacted by LC fractionation depth, MS isolation window, and peptide loading amount. Exhaustive fractionation (320 × 4 h) can nearly eliminate the interference and achieve results comparable to the MS3-based method. Importantly, the interference in MS2 scans can be estimated by the intensity of contaminated y1 product ions, and we thus developed an algorithm to correct reporter ion ratios of tryptic peptides. Our data indicate that intermediate fractionation (40 × 2 h) and y1 ion-based correction allow accurate and deep TMT profiling of more than 10 000 proteins, which represents a straightforward and affordable strategy in isobaric labeling proteomics.

Extensive Peptide Fractionation and y1 Ion-Based Interference Detection Method for Enabling Accurate Quantification by Isobaric Labeling and Mass Spectrometry.

The molecular circuits by which antigens activate quiescent T cells remain poorly understood. We combined temporal profiling of the whole proteome and phosphoproteome via multiplexed isobaric labeling proteomics technology, computational pipelines for integrating multi-omics datasets, and functional perturbation to systemically reconstruct regulatory networks underlying T cell activation. T cell receptors activated the T cell proteome and phosphoproteome with discrete kinetics, marked by early dynamics of phosphorylation and delayed ribosome biogenesis and mitochondrial activation. Systems biology analyses identified multiple functional modules, active kinases, transcription factors and connectivity between them, and mitochondrial pathways including mitoribosomes and complex IV. Genetic perturbation revealed physiological roles for mitochondrial enzyme COX10-mediated oxidative phosphorylation in T cell quiescence exit. Our multi-layer proteomics profiling, integrative network analysis, and functional studies define landscapes of the T cell proteome and phosphoproteome and reveal signaling and bioenergetics pathways that mediate lymphocyte exit from quiescence.

Integrative Proteomics and Phosphoproteomics Profiling Reveals Dynamic Signaling Networks and Bioenergetics Pathways Underlying T Cell Activation.

N-terminal acetylation is an abundant modification influencing protein functions. Because ∼80% of mammalian cytosolic proteins are N-terminally acetylated, this modification is potentially an untapped target for chemical control of their functions. Structural studies have revealed that, like lysine acetylation, N-terminal acetylation converts a positively charged amine into a hydrophobic handle that mediates protein interactions; hence, this modification may be a druggable target. We report the development of chemical probes targeting the N-terminal acetylation-dependent interaction between an E2 conjugating enzyme (UBE2M or UBC12) and DCN1 (DCUN1D1), a subunit of a multiprotein E3 ligase for the ubiquitin-like protein NEDD8. The inhibitors are highly selective with respect to other protein acetyl-amide-binding sites, inhibit NEDD8 ligation in vitro and in cells, and suppress anchorage-independent growth of a cell line with DCN1 amplification. Overall, our data demonstrate that N-terminal acetyl-dependent protein interactions are druggable targets and provide insights into targeting multiprotein E2-E3 ligases.

Article	Year
Synaptic protein ubiquitination in rat brain revealed by antibody-based ubiquitome analysis. Journal of proteome research\| PubMed ID: 22871113	2012
U1 small nuclear ribonucleoprotein complex and RNA splicing alterations in Alzheimer's disease. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 24023061	2013
Functionally enigmatic genes: a case study of the brain ignorome. PloS one\| PubMed ID: 24523945	2014
JUMP: a tag-based database search tool for peptide identification with high sensitivity and accuracy. Molecular & cellular proteomics : MCP\| PubMed ID: 25202125	2014
Refined phosphopeptide enrichment by phosphate additive and the analysis of human brain phosphoproteome. Proteomics\| PubMed ID: 25307156	2015
Systematic optimization of long gradient chromatography mass spectrometry for deep analysis of brain proteome. Journal of proteome research\| PubMed ID: 25455107	2015
Asymmetrical macromolecular complex formation of lysophosphatidic acid receptor 2 (LPA2) mediates gradient sensing in fibroblasts. The Journal of biological chemistry\| PubMed ID: 25542932	2014
Quantitative protein analysis by mass spectrometry. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 25859956	2015
Sequential Elution Interactome Analysis of the Mind Bomb 1 Ubiquitin Ligase Reveals a Novel Role in Dendritic Spine Outgrowth. Molecular & cellular proteomics : MCP\| PubMed ID: 25931508	2015
Altered cGMP dynamics at the plasma membrane contribute to diarrhea in ulcerative colitis. The American journal of pathology\| PubMed ID: 26261085	2015
Joint mouse-human phenome-wide association to test gene function and disease risk. Nature communications\| PubMed ID: 26833085	2016
JUMPg: An Integrative Proteogenomics Pipeline Identifying Unannotated Proteins in Human Brain and Cancer Cells. Journal of proteome research\| PubMed ID: 27225868	2016
Extensive Peptide Fractionation and y1 Ion-Based Interference Detection Method for Enabling Accurate Quantification by Isobaric Labeling and Mass Spectrometry. Analytical chemistry\| PubMed ID: 28194965	2017
Integrative Proteomics and Phosphoproteomics Profiling Reveals Dynamic Signaling Networks and Bioenergetics Pathways Underlying T Cell Activation. Immunity\| PubMed ID: 28285833	2017
Blocking an N-terminal acetylation-dependent protein interaction inhibits an E3 ligase. Nature chemical biology\| PubMed ID: 28581483	2017

Xusheng Wang

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