Complex Proteomes Research Articles

Bioorthogonal Chemical Probes of Lysine Acetyltransferases

Acetylation of lysine residues is one of the most important posttranslational modifications that diversify protein functions. Thousands of acetylated lysine residues have been identified, suggesting that the effects of lysine acetyltransferases (KATs) go far beyond the chromatin kingdom. However, there is a missing link connecting the compositions of the cellular acetylome networks to the enzymatic activities of different KAT members. The outstanding challenge is how to dissect the subacetylomes of individual KATs and address their functions on the proteomic scale. We have explored a bioorthogonal profiling of protein acetylation strategy to label and interrogate substrates of KAT enzymes. In this strategy, a suite of Ac‐CoA analogs containing either alkynyl or azido functional group (e.g. 3AZ‐CoA, 4AZ‐CoA, 4PY‐CoA, 5HY‐CoA, 6HY‐CoA) were synthesized as potential cofactor surrogate for selective labeling of KAT substrates. Meanwhile, the active site of the KATs was engineered in order to expand the cofactor binding capability of the enzymes to accommodate the bulkier synthetic cofactors. The acylated substrates can be selectively linked through the copper‐catalyzed azide‐alkyne cycloaddition (CuAAC) reaction with fluorescent reporter or biotin affinity tag for optical imaging or protein enrichment on streptavidin‐coated resin. We have successfully used this bioorthogonal technology to profile substrates of p300 and GCN5 in the context of complex cellular proteomes. Together with LC‐MS/MS technology, more than four hundred proteins were identified as GCN5 or p300 substrate candidates. These proteins are extensively involved in various biological events including gene expression, cell cycle, and cellular metabolism. Overall, we have invented a bioorthogonal, chemoproteomic technology to investigate KAT biology. This chemical biology strategy provides a powerful enabling technology for activity‐based lysine acylation profiling on proteomic scale. Indeed, our KAT activity profiling demonstrates extensive engagement of KATs in cellular pathways and provides new molecular insights into understanding their functions in epigenetics and other biological processes.bioorthogonal profilingFigure 1

Rapid and Quantitative Protein Precipitation for Proteome Analysis by Mass Spectrometry.

Protein precipitation is a common front-end preparation strategy for proteome analysis, as well as other applications (e.g., protein depletion for small molecule analysis, bulk commercial preparation of protein). Highly variable conditions used to precipitate proteins, ranging in solvent type, strength, time, and temperature, reflect inconsistent and low recovery. As a consequence, incomplete proteome coverage diminishes the utility of precipitation for proteome sample preparation ahead of mass spectrometry. We herein investigate and optimize the conditions affecting protein recovery through precipitation using acetone at a defined ionic strength. By increasing the salt concentration and incubation temperature with 80% acetone, we show that rapid (2 min) precipitation provides consistently high protein recovery (98 ± 1%) of complex proteome extracts. Rapid precipitation is also applicable to isolate dilute proteins starting as low as 1 μg mL-1. Furthermore, analysis of the protein pellet by bottom-up mass spectrometry (MS) reveals unbiased recovery of all proteins with respect to molecular weight, isoelectric point (pI), and hydrophobicity. Our robust strategy to isolate proteins maximizes recovery and throughput, exploiting the analytical advantages of precipitation over alternative techniques. Data are available via ProteomeXchange with identifier PXD015674.

An experimentally generated peptide database increases the sensitivity of XL-MS with complex samples

Cross-linking mass spectrometry (XL-MS) is steadily expanding its range of applications from purified protein complexes to more complex samples like organelles and even entire cells. One main challenge using non-cleavable cross-linkers is the so-called n2 problem: With linearly increasing database size, the search space for the identification of two covalently linked peptides per spectrum increases quadratically. Here, we report an alternative search strategy that focuses on only those peptides, which were demonstrated to cross-link under the applied experimental conditions. The performance of a parallel XL-MS experiment using a thiol-cleavable cross-linker enabled the identification of peptides that carried a cleaved cross-link moiety after reduction and hence were involved in cross-linking reactions. Based on these identifications, a peptide database was generated and used for the database search of the actual cross-linking experiment with a non-cleavable cross-linker. This peptide-focused approach was tested on protein complexes with a reported structural model and obtained results corresponded well to a conventional database search. An application of the strategy on in vivo cross-linked Bacillus subtilis and Bacillus cereus cells revealed a five- to tenfold reduction in search time and led to significantly more identifications with the latter species than a search against the entire proteome. SignificanceInstead of considering all theoretically cross-linkable peptides in a proteome, identification and pre-filtering for a subset of cross-link peptide candidates allows for a dramatically decreased search space. Hence, there is less potential for the random accumulation of false positives ultimately leading to a higher sensitivity in the XL-MS experiment. Using the peptide-focused approach, a cross-linking database search can be conducted in a fraction of time while yielding a similar or higher number of identifications, thereby enabling the cross-linking analysis of samples of mammalian proteome complexity.

Enrichment of Cysteine-Containing Peptide by On-Resin Capturing and Fixed Charge Tag Derivatization for Sensitive ESI-MS Detection.

High complexity of cell and tissue proteomes limits the investigation of proteomic biomarkers. Therefore, the methods of enrichment of some chemical groups of peptides including thiopeptides are important tools that may facilitate the proteomic analysis by reducing sample complexity and increasing proteome coverage. Here, we present a new method of cysteine-containing tryptic peptide enrichment using commercially available TentaGel R RAM resin modified by the linker containing the maleimide group, allowing thiol conjugation. The captured tryptic peptides containing lysine residue were then tagged by 2,4,6-triphenylpyrylium salt to form 2,4,6-triphenylpyridinium derivatives, which increases the ionization efficiency during mass spectrometry analysis. This makes it possible to conduct an ultrasensitive analysis of the trace amount of compounds. The proposed strategy was successfully applied in the enrichment of model tryptic podocin peptide and podocin tryptic digest.

3-Oxo-β-sultam as a Sulfonylating Chemotype for Inhibition of Serine Hydrolases and Activity-Based Protein Profiling.

3-Oxo-β-sultams are four-membered ring ambident electrophiles that can react with nucleophiles either at the carbonyl carbon or at the sulfonyl sulfur atoms, and that have been reported to inhibit serine hydrolases via acylation of the active-site serine residue. We have developed a panel of 3-oxo-β-sultam inhibitors and show, through crystallographic data, that they are regioselective sulfonylating electrophiles, covalently binding to the catalytic serine of human and porcine elastases through the sulfur atom. Application of 3-oxo-β-sultam-derived activity-based probes in a human proteome revealed their potential to label disease-related serine hydrolases and proteasome subunits. Activity-based protein profiling applications of 3-oxo-β-sultams should open up new opportunities to investigate these classes of enzymes in complex proteomes and expand the toolbox of available sulfur-based covalent protein modifiers in chemical biology.

High-Throughput Identification of MHC Class I Binding Peptides Using an Ultradense Peptide Array.

Rational vaccine development and evaluation requires identifying and measuring the magnitude of epitope-specific CD8 T cell responses. However, conventional CD8 T cell epitope discovery methods are labor intensive and do not scale well. In this study, we accelerate this process by using an ultradense peptide array as a high-throughput tool for screening peptides to identify putative novel epitopes. In a single experiment, we directly assess the binding of four common Indian rhesus macaque MHC class I molecules (Mamu-A1*001, -A1*002, -B*008, and -B*017) to ∼61,000 8-mer, 9-mer, and 10-mer peptides derived from the full proteomes of 82 SIV and simian HIV isolates. Many epitope-specific CD8 T cell responses restricted by these four MHC molecules have already been identified in SIVmac239, providing an ideal dataset for validating the array; up to 64% of these known epitopes are found in the top 192 SIVmac239 peptides with the most intense MHC binding signals in our experiment. To assess whether the peptide array identified putative novel CD8 T cell epitopes, we validated the method by IFN-γ ELISPOT assay and found three novel peptides that induced CD8 T cell responses in at least two Mamu-A1*001-positive animals; two of these were validated by ex vivo tetramer staining. This high-throughput identification of peptides that bind class I MHC will enable more efficient CD8 T cell response profiling for vaccine development, particularly for pathogens with complex proteomes for which few epitope-specific responses have been defined.

Full-Featured, Real-Time Database Searching Platform Enables Fast and Accurate Multiplexed Quantitative Proteomics.

Multiplexed quantitative analyses of complex proteomes enable deep biological insight. While a multitude of workflows have been developed for multiplexed analyses, the most quantitatively accurate method (SPS-MS3) suffers from long acquisition duty cycles. We built a new, real-time database search (RTS) platform, Orbiter, to combat the SPS-MS3 method's longer duty cycles. RTS with Orbiter eliminates SPS-MS3 scans if no peptide matches to a given spectrum. With Orbiter's online proteomic analytical pipeline, which includes RTS and false discovery rate analysis, it was possible to process a single spectrum database search in less than 10 ms. The result is a fast, functional means to identify peptide spectral matches using Comet, filter these matches, and more efficiently quantify proteins of interest. Importantly, the use of Comet for peptide spectral matching allowed for a fully featured search, including analysis of post-translational modifications, with well-known and extensively validated scoring. These data could then be used to trigger subsequent scans in an adaptive and flexible manner. In this work we tested the utility of this adaptive data acquisition platform to improve the efficiency and accuracy of multiplexed quantitative experiments. We found that RTS enabled a 2-fold increase in mass spectrometric data acquisition efficiency. Orbiter's RTS quantified more than 8000 proteins across 10 proteomes in half the time of an SPS-MS3 analysis (18 h for RTS, 36 h for SPS-MS3).

Enhancing protein discoverability by data independent acquisition assisted by ion mobility mass spectrometry

Ion mobility (IM) mass spectrometry allows conducting data independent acquisition (DIA) where all ions entering the instrument are fragmented based on their drift time. In this work, DIA operational parameters were first optimized using a design of experiments. The optimization of data treatment involved a smoothing algorithm of the IM dimension, which increased the number of identified peptides. Then, classical DDA and IM-based DIA were compared injecting increasing amounts of a complex proteome digest (E. coli). Results revealed that compared to DDA, DIA allowed to identify from 2 to 3.3 times more proteins, depending on the injected quantity. To evaluate proteome coverage, endogenous proteins in E. coli cells were sorted by abundance deciles. A large majority of the proteins uniquely observed in DDA were part of the 10% most abundant protein groups. Interestingly, owing to the absence of ion-picking algorithm, DIA allowed to identify proteins coming from a broader concentration range therefore greatly improving proteome coverage. Furthermore, ion mobility separation improved coverage by separating co-eluting peptides. Physicochemical properties of peptides uniquely detected by DIA or DDA were also compared using supervised and unsupervised multivariate analysis. As a result, peptides having a higher mass and being relatively hydrophobic were significantly more identified in DIA. Finally, semi-quantitative performance of both methods was investigated and proved to be comparable, except that DIA demonstrated a better sensitivity than DDA. As a conclusion, we demonstrated in this study that both acquisition modes provide complementary information about the proteome under investigation.

Probing the Refoldability of the Proteome with Mass Spectrometry

Decades of research on protein folding have primarily focused on a privileged subset of small, single domain proteins that can reversibly refold out of a denaturant. However, these studies are not representative of the complexity of natural proteomes, which consist of many proteins with more sophisticated architectures and topologies. Here, we introduce a novel experimental approach to probe the kinetics of protein folding on the scale of whole proteomes. We accomplish this by first unfolding and refolding whole lysates, and then probing the resulting protein structures using a permissive protease that preferentially cleaves at flexible regions.

Post-Transcriptional Regulation of Homeostatic, Stressed, and Malignant Stem Cells.

Cellular identity is not driven by differences in genomic content but rather by epigenomic, transcriptomic, and proteomic heterogeneity. Although regulation of the epigenome plays a key role in shaping stem cell hierarchies, differential expression of transcripts only partially explains protein abundance. The epitranscriptome, translational control, and protein degradation have emerged as fundamental regulators of proteome complexity that regulate stem cell identity and function. Here, we discuss how post-transcriptional mechanisms enable stem cell homeostasis and responsiveness to developmental cues and environmental stressors by rapidly shaping the content of their proteome and how these processes are disrupted in pre-malignant and malignant states.

DIALib: an automated ion library generator for data independent acquisition mass spectrometry analysis of peptides and glycopeptides.

Data Independent Acquisition (DIA) Mass Spectrometry (MS) workflows allow unbiased measurement of all detectable peptides from complex proteomes, but require ion libraries for interrogation of peptides of interest. These DIA ion libraries can be theoretical or built from peptide identification data from Data Dependent Acquisition (DDA) MS workflows. However, DDA libraries derived from empirical data rely on confident peptide identification, which can be challenging for peptides carrying complex post-translational modifications. Here, we present DIALib, software to automate the construction of peptide and glycopeptide Data Independent Acquisition ion Libraries. We show that DIALib theoretical ion libraries can identify and measure diverse N- and O-glycopeptides from yeast and mammalian glycoproteins without prior knowledge of the glycan structures present. We present proof-of-principle data from a moderately complex yeast cell wall glycoproteome and a simple mixture of mammalian glycoproteins. We also show that DIALib libraries consisting only of glycan oxonium ions can quickly and easily provide a global compositional glycosylation profile of the detectable "oxoniome" of glycoproteomes. DIALib will help enable DIA glycoproteomics as a complementary analytical approach to DDA glycoproteomics.

A Protocol for the Plasma Membrane Proteome Analysis of Rice Leaves.

Subcellular proteome analysis is one of the most effective ways to reduce the complexity of total proteome. With the advancement in protein extraction methodologies, it is now possible to fractionate and isolate the proteins from subcellular compartments without significant contamination from the cytoplasm and other organelles. Of the different subcellular proteomes, plasma membrane remained largely uncharacterized because of the difficulties in isolation of contamination free plasma membrane proteins. Moreover, proteome analysis in the past two decades majorly relied on the two-dimensional gel electrophoresis which showed limited protein loading ability and poor separation of highly hydrophobic plasma membrane proteins. Development of shotgun proteomics methods has facilitated the identification and quantification of hydrophobic proteins isolated from plasma membrane or other cellular membranes. Here, we present a simplified procedure for the isolation of plasma membrane proteins by a two-phase partitioning method and their identification by shotgun proteomics approach using rice as a model plant.

Subcellular Proteomics in Conifers: Purification of Nuclei and Chloroplast Proteomes.

The complexity of the plant cell proteome, exhibiting thousands of proteins whose abundance varies in several orders of magnitude, makes impossible to cover most of the plant proteins using standard shotgun-based approaches. Despite this general description of plant proteomes, the complexity is not a big issue (current protocols and instrumentation allow for the identification of several thousand proteins per injection), low or medium abundant proteins cannot be detected most of times, being necessary to fraction or perform targeted analyses in order to detect and quantify them. Among fractioning choices, cell fractioning in its different organelles is a good strategy for gaining not only a deeper coverage of the proteome but also the basis for understanding organelle function, protein dynamics, and trafficking within the cell, as nuclear and chloroplast communication. This approach is used routinely in many labs working with model species; however, the available protocols focusing on tree species are scarce. In this chapter, we provide a simple but robust protocol for isolating nuclei and chloroplasts in pine needles that is fully compatible with later mass spectrometry-based proteome analysis.

Chromatin-sensitive cryptic promoters putatively drive expression of alternative protein isoforms in yeast.

Cryptic transcription is widespread and generates a heterogeneous group of RNA molecules of unknown function. To improve our understanding of cryptic transcription, we investigated their transcription start site (TSS) usage, chromatin organization, and posttranscriptional consequences in Saccharomyces cerevisiae. We show that TSSs of chromatin-sensitive internal cryptic transcripts retain comparable features of canonical TSSs in terms of DNA sequence, directionality, and chromatin accessibility. We define the 5′ and 3′ boundaries of cryptic transcripts and show that, contrary to RNA degradation–sensitive ones, they often overlap with the end of the gene, thereby using the canonical polyadenylation site, and associate to polyribosomes. We show that chromatin-sensitive cryptic transcripts can be recognized by ribosomes and may produce truncated polypeptides from downstream, in-frame start codons. Finally, we confirm the presence of the predicted polypeptides by reanalyzing N-terminal proteomic data sets. Our work suggests that a fraction of chromatin-sensitive internal cryptic promoters initiates the transcription of alternative truncated mRNA isoforms. The expression of these chromatin-sensitive isoforms is conserved from yeast to human, expanding the functional consequences of cryptic transcription and proteome complexity.

Deep profiling and custom databases improve detection of proteoforms generated by alternative splicing.

Alternative pre-mRNA splicing has long been proposed to contribute greatly to proteome complexity. However, the extent to which mature mRNA isoforms are successfully translated into protein remains controversial. Here, we used high-throughput RNA sequencing and mass spectrometry (MS)–based proteomics to better evaluate the translation of alternatively spliced mRNAs. To increase proteome coverage and improve protein quantitation, we optimized cell fractionation and sample processing steps at both the protein and peptide level. Furthermore, we generated a custom peptide database trained on analysis of RNA-seq data with MAJIQ, an algorithm optimized to detect and quantify differential and unannotated splice junction usage. We matched tandem mass spectra acquired by data-dependent acquisition (DDA) against our custom RNA-seq based database, as well as SWISS-PROT and RefSeq databases to improve identification of splicing-derived proteoforms by 28% compared with use of the SWISS-PROT database alone. Altogether, we identified peptide evidence for 554 alternate proteoforms corresponding to 274 genes. Our increased depth and detection of proteins also allowed us to track changes in the transcriptome and proteome induced by T-cell stimulation, as well as fluctuations in protein subcellular localization. In sum, our data here confirm that use of generic databases in proteomic studies underestimates the number of spliced mRNA isoforms that are translated into protein and provides a workflow that improves isoform detection in large-scale proteomic experiments.

Comparative analysis of differential proteome-wide protein-protein interaction network of Methanobrevibacter ruminantium M1

A proteome-wide protein-protein interaction (PPI) network of Methanobrevibacter ruminantium M1 (MRU), a predominant rumen methanogen, was constructed from its metabolic genes using a gene neighborhood algorithm and then compared with closely related rumen methanogens Using proteome-wide PPI approach, we constructed network encompassed 2194 edges and 637 nodes interacting with 634 genes. Network quality and robustness of functional modules were assessed with gene ontology terms. A structure-function-metabolism mapping for each protein has been carried out with efforts to extract experimental PPI concomitant information from the literature. The results of our study revealed that some topological properties of its network were robust for sharing homologous protein interactions across heterotrophic and hydrogenotrophic methanogens. MRU proteome has shown to establish many PPI sub-networks for associated metabolic subsystems required to survive in the rumen environment. MRU genome found to share interacting proteins from its PPI network involved in specific metabolic subsystems distinct to heterotrophic and hydrogenotrophic methanogens. Across these proteomes, the interacting proteins from differential PPI networks were shared in common for the biosynthesis of amino acids, nucleosides, and nucleotides and energy metabolism in which more fractions of protein pairs shared with Methanosarcina acetivorans. Our comparative study expedites our knowledge to understand a complex proteome network associated with typical metabolic subsystems of MRU and to improve its genome-scale reconstruction in the future.

Innovating the Concept and Practice of Two-Dimensional Gel Electrophoresis in the Analysis of Proteomes at the Proteoform Level.

Two-dimensional gel electrophoresis (2DE) is an important and well-established technical platform enabling extensive top-down proteomic analysis. However, the long-held but now largely outdated conventional concepts of 2DE have clearly impacted its application to in-depth investigations of proteomes at the level of protein species/proteoforms. It is time to popularize a new concept of 2DE for proteomics. With the development and enrichment of the proteome concept, any given “protein” is now recognized to consist of a series of proteoforms. Thus, it is the proteoform, rather than the canonical protein, that is the basic unit of a proteome, and each proteoform has a specific isoelectric point (pI) and relative mass (Mr). Accordingly, using 2DE, each proteoform can routinely be resolved and arrayed according to its different pI and Mr. Each detectable spot contains multiple proteoforms derived from the same gene, as well as from different genes. Proteoforms derived from the same gene are distributed into different spots in a 2DE pattern. High-resolution 2DE is thus actually an initial level of separation to address proteome complexity and is effectively a pre-fractionation method prior to analysis using mass spectrometry (MS). Furthermore, stable isotope-labeled 2DE coupled with high-sensitivity liquid chromatography-tandem MS (LC-MS/MS) has tremendous potential for the large-scale detection, identification, and quantification of the proteoforms that constitute proteomes.

Comparative analysis of whey proteins in donkey colostrum and mature milk using quantitative proteomics.

Donkey milk is attracting increasing attention as a nutritional milk source similar to human milk. In this study, we carried out qualitative and quantitative analysis of the donkey whey proteome using a label-free proteomic approach, combined with parallel reaction monitoring (PRM) as a validation method. A total of 300 whey proteins were identified in donkey colostrum (DC) and donkey mature (DM) milk, of which 18 were differentially expressed (P < 0.05) between the two types of milk. Gene ontology (GO) analysis showed that differentially and uniquely expressed proteins were mainly involved in cellular processes, response to stimulus, metabolic processes, and biological regulation. Their molecular functions included binding, catalytic activity, and molecular functional regulation, and their main annotated areas of origin were the cell, cell-part, and the extracellular region. Most differentially and uniquely expressed proteins were linked with malaria, systemic lupus erythematosus, or antigen processing and presentation. Our results provide insight into the complexity of the donkey whey proteome and molecular evidence for nutritional differences between different lactation stages.

A novel approach for protein identification from complex cell proteome using modified peptide mass fingerprinting algorithm.

A unique peptide based search algorithm for identification of protein mixture using PMF is proposed. The proposed search algorithm utilizes binary search and heapsort programs to generate frequency chart depicting the unique peptides corresponding to all proteins in a proteome. The use of binary search program significantly reduces the time for frequency chart preparation to ∼2 s for a proteome comprising ∼23000 proteins. The algorithm was applied to a three-protein mixture identification, host cell protein (HCP) analysis, and a simulation-generated data set. It was found that the algorithm could identify at least one unique peptide of a protein even in the presence of fourfold higher concentration of another protein. In addition, two HCPs that are known to be difficult to remove were missed by MS/MS approach and were exclusively identified using the presented algorithm. Thus, the proposed algorithm when used along with standard proteomic approaches present avenues for enhanced protein identification efficiency, particularly for applications such as HCP analysis in biopharmaceutical research, where identification of low-abundance proteins are generally not achieved due to dynamic range limitations between the target product and HCPs.

Differential Expression of MARK4 Protein and Related Perturbations in Females with Ovulatory PCOS.

Ovulatory PCOS (OPCOS) is the mildest form of the polycystic ovarian syndrome among all four determined phenotypes. Though the females with OPCOS are ovulating, hyperandrogenism and polycystic ovarian morphology increase the susceptibility of cardiovascular diseases, insulin resistance, hyperlipidemia and metabolic syndrome in these females. The aim of the study was to identify the significance associated with OPCOS phenotype through serum proteomic profiling of OPCOS females and normal age-matched healthy ovulating females. One and two-dimensional gel-based proteomic approaches were adopted to fractionate the complex serum proteome. Differential protein profiles generated were analyzed with PD-QUEST Software. Protein spots differing in intensity by >2-fold were selected and identified further by MALDI-TOF MS. Validation of identified protein was carried out by Biolayer Interferometry. One and two-dimensional gel profiles revealed a differential expression pattern of proteins. 10 selected spots were identified as GMP synthase [glutamine hydrolyzing], zinc finger protein 518A, pericentriolar material 1 protein, BCLAF1 and THRAP3 family member 3, MAP/microtubule affinityregulating kinase 4, H/ACA ribonucleoprotein complex subunit 1, Melanoma-associated antigen B3 and Zinc finger protein 658B. Expression of MAP/microtubule affinity-regulating kinase 4 (MARK4) was found to be downregulated in OPCOS females as compared to controls on validation. Reduced expression of MARK4 protein in OPCOS increases the associated risk of hyperlipidemia, hyperandrogenism and metabolic syndrome, thus the protein holds strong candidature as a drug target for the syndrome.