Complementary Mass Spectrometry Research Articles

Single particle cryo-EM of the complex between interphotoreceptor retinoid-binding protein and a monoclonal antibody.

Interphotoreceptor retinoid‐binding protein (IRBP) is a highly expressed protein secreted by rod and cone photoreceptors that has major roles in photoreceptor homeostasis as well as retinoid and polyunsaturated fatty acid transport between the neural retina and retinal pigment epithelium. Despite two crystal structures reported on fragments of IRBP and decades of research, the overall structure of IRBP and function within the visual cycle remain unsolved. Here, we studied the structure of native bovine IRBP in complex with a monoclonal antibody (mAb5) by cryo‐electron microscopy, revealing the tertiary and quaternary structure at sufficient resolution to clearly identify the complex components. Complementary mass spectrometry experiments revealed the structure and locations of N‐linked carbohydrate post‐translational modifications. This work provides insight into the structure of IRBP, displaying an elongated, flexible three‐dimensional architecture not seen among other retinoid‐binding proteins. This work is the first step in elucidation of the function of this enigmatic protein.

Quantitation and localization of beta-blockers and SSRIs accumulation in fathead minnows by complementary mass spectrometry analyses

Beta-blockers and selective serotonin reuptake inhibitors (SSRIs) enter the environment through the improper disposal of drugs in garbage, sinks, or toilets as well as via excretion after their intended use. Beta-blockers and SSRIs are resistant to biodegradation and highly water soluble, leading to limited removal capabilities by wastewater treatment plants. As a result, these compounds can easily enter the aquatic environment in a biologically active state, posing risks to the behavior, anatomy, and physiology of aquatic organisms through exposure. However, there is insufficient research on the effects of beta-blockers on aquatic vertebrates to have a complete understanding of the impact of beta-blocker pollution on aquatic organisms. Exposing fathead minnows at environmentally-relevant concentrations allows for an accurate representation of how beta-blockers and SSRIs affect and accumulate in aquatic vertebrates. Singular exposures to two beta-blockers and two selective serotonin reuptake inhibitors (SSRIs) at seven days modeled how pollution affects aquatic wildlife during acute exposure events. After exposure, the entire organism was analyzed quantitatively by liquid chromatography mass spectrometry and qualitatively by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). Imaging allowed for the determination of the part of the organism in which the drugs were accumulating. Results showed that after seven days of exposure there was accumulation of both the beta-blockers and SSRIs in the fathead minnows. This data represents the first application of LC-MS quantitation guided by MALDI-MSI in aquatic toxicology-type research in environmental analytical chemistry.

Phloem sap in Cretaceous ambers as abundant double emulsions preserving organic and inorganic residues

Fossilized remains preserved in amber provide abundant data on the paleobiota surrounding the resin-producing plants, but relatively scarcer information about the resinous sources themselves. Here, dark pseudoinclusions in kidney-shaped amber pieces from the Early Cretaceous (Albian) amber from Spain are studied. This type of fossilized remain, abundant in Cretaceous ambers, was first interpreted as fossilized vacuole-bearing microorganisms, but later regarded as artifactual and probably secreted by the resinous trees, although their origin remained unclear. Using complementary microscopy (light, electron, confocal), spectroscopy (infrared, micro-Raman), mass spectrometry and elemental analysis techniques, we demonstrate that the pseudoinclusions correspond to droplets of phloem sap containing amber spheroids and preserving both organic and inorganic residues consistent with degraded components from the original sap. The amber pieces containing pseudoinclusions are fossilized, resin-in-sap-in-resin double emulsions, showing banding patterns with differential content of resin-in-sap emulsion droplets. Our findings represent the first time fossilized phloem sap, 105 million years old, has been recognized and characterized, and open new lines of paleontological research with taxonomic, taphonomic, physiological and ecological implications.

Accelerated instability testing reveals quantitative mass spectrometry overcomes specimen storage limitations associated with PD-L1 immunohistochemistry

Immunohistochemistry (IHC) using formalin-fixed, paraffin embedded (FFPE) tissue is limited by epitope masking, posttranslational modification and immunoreactivity loss that occurs in stored tissue by poorly characterized mechanisms. Conformational epitopes recognized by many programmed-death-ligand-1 (PD-L1) IHC assays are particularly susceptible to degradation and provide an ideal model for understanding signal loss in stored FFPE tissue. Here we assessed 1206 tissue sections to evaluate environmental factors impacting immunoreactivity loss. PD-L1 IHC using four antibodies (22C3, 28-8, E1L3N, and SP142), raised against intracellular and extracellular epitopes, was assessed in stored FFPE tissue alongside quantitative mass spectrometry (MS). Global proteome analyses were used to assess proteome-wide oxidation across an inventory of 3041 protein groups (24,737 distinct peptides). PD-L1 quantitation correlated well with IHC expression on unaged sections (R2 = 0.744; P < 0.001), with MS demonstrating no loss of PD-L1 protein, even in sections with significant signal loss by IHC impacting diagnostic category. Clones 22C3 and 28-8 were most susceptible to signal loss, with E1L3N demonstrating the most robust signal (56%, 58%, and 33% reduction respectively; p < 0.05). Increased humidity and temperature resulted in significant acceleration of immunoreactivity loss, which was mitigated by storage with desiccant. MS demonstrated only modest oxidation of 274 methionine-containing peptides and aligned with IHC results suggesting peptide oxidation is not a major factor. These data imply immunoreactivity loss driven by humidity and temperature results in structural distortion of epitopes rendering them unsuitable for antibody binding following epitope retrieval. Limitations of IHC biomarker analysis from stored tissue sections may be mitigated by cost-effective use of desiccant when appropriate. In some scenarios, complementary MS is a preferred approach for retrospective analyses of archival FFPE tissue collections.

Characterization of Steroids through Collision Cross Sections: Contribution of Quantum Chemistry Calculations.

A wide range of collision cross section (CCS) databases for different families of compounds have recently been established from ion mobility mass spectrometry (IM-MS) measurements. Nevertheless, the need to validate these new data sets to provide the necessary confidence about the use of this parameter is increasingly expressed by the scientific community. If such a validation requires that complementary mass spectrometry experiments are conducted, it also appears that alternative strategies can contribute to the validation of such empirical data. In particular, in silico approaches are relevant to compute theoretical CCS values, to be compared to experimental ones. A recently published CCS database for 300 steroids allowed one to observe experimentally significant deviations of the expected CCS versus m/z correlations for some compounds. The present work attempts to rationalize such deviations with Density Functional Theory (DFT) calculations. MN15/6-311++G(d,p) investigations have been carried out, starting with a conformational analysis of a sample of 20 selected steroids and the determination of their preferred gas-phase ionization site. CCS values were then computed and compared to the experimental data. This approach allowed one to rationalize the experimental trends, providing an accurate description of the key properties of the various steroids considered.

Kinetic and structural characterization of therapeutic albumin chemical functionalization using complementary mass spectrometry techniques

Protein conjugates such as antibody-drugs conjugates (ADCs) represents the next generation of therapeutic proteins. They allow to combine the biological properties of the protein format with the characteristics of the conjugated ligands. The reaction implemented to couple ligands to the peptide backbone represents a crucial aspect of the production of protein conjugates, influencing the nature and the heterogeneity of the conjugates obtained. Here, we report the concomitant use of MALDI-TOF MS and LC–MS/MS analysis to investigate the chemical functionalization of human serum albumin (HSA) by the intermediate of lysine residues, previously used to generate biopharmaceutical agents for medical imaging. A kinetic was performed by collecting samples after different reaction times and analyzing them using the two techniques. MALDI-TOF MS analyses allowed estimating the number of conjugated ligands in a robust manner and assess the global functionalization kinetic on the intact protein level. Results demonstrated a maximum of 38 modified residues out of the 59 lysines available showing the limitation of the chemical functionalization. Consequently, LC–MS/MS analysis provided a site-specific characterization of the residues undergoing chemical modification. Data exhibited unique properties due to the presence of the ligands which allowed to identify without ambiguity the residues exhibiting different modification rate and enabled the identification of the unmodified lysine. Results were compared to the structure of HSA described from crystallography data. The comparison strongly suggested that accessibility is influencing the residues respective reactivity. The relevant complementarity of the different techniques could be emphasized in order to perform an extensive characterization concerning the evolution of the primary structure of the protein during the chemical reaction, providing an improved insight on the conjugation process and offering the potentiality to tune the reaction.

Toward Improved Accuracy in Chlorine Isotope Analysis: Synthesis Routes for In-House Standards and Characterization via Complementary Mass Spectrometry Methods.

Increasing applications of compound-specific chlorine isotope analysis (CSIA) emphasize the need for chlorine isotope standards that bracket a wider range of isotope values in order to ensure accurate results. With one exception (USGS38), however, all international chlorine isotope reference materials (chloride and perchlorate salts) fall within the narrow range of one per mille. Furthermore, compound-specific working standards are required for chlorine CSIA but are not available for most organic substances. We took advantage of isotope effects in chemical dehalogenation reactions to generate (i) silver chloride (CT16) depleted in 37Cl/35Cl and (ii) compound-specific standards of the herbicides acetochlor and S-metolachlor (Aceto2, Metola2) enriched in 37Cl/35Cl. Calibration against the international reference standards USGS38 (-87.90 ‰) and ISL-354 (+0.05 ‰) by complementary methods (gas chromatography-isotope ratio mass spectrometry, GC-IRMS, versus gas chromatography-multicollector inductively coupled plasma mass spectrometry, GC-MC-ICPMS) gave a consensus value of δ37ClCT16 = -26.82 ± 0.18 ‰. Preliminary GC-MC-ICPMS characterization of commercial Aceto1 and Metola1 versus Aceto2 and Metola2 resulted in tentative values of δ37ClAceto1 = 0.29 ± 0.29 ‰, δ37ClAceto2 = 18.54 ± 0.20 ‰, δ37ClMetola1 = -4.28 ± 0.17 ‰ and δ37ClMetola2 = 5.12 ± 0.27 ‰. The possibility to generate chlorine isotope in-house standards with pronounced shifts in isotope values offers a much-needed basis for accurate chlorine CSIA.

Nuclear actin interactome analysis links actin to KAT14 histone acetyl transferase and mRNA splicing.

ABSTRACTIn addition to its essential functions within the cytoskeleton, actin also localizes to the cell nucleus, where it is linked to many important nuclear processes from gene expression to maintenance of genomic integrity. However, the molecular mechanisms by which actin operates in the nucleus remain poorly understood. Here, we have used two complementary mass spectrometry (MS) techniques, AP-MS and BioID, to identify binding partners for nuclear actin. Common high-confidence interactions highlight the role of actin in chromatin-remodeling complexes and identify the histone-modifying complex human Ada-Two-A-containing (hATAC) as a novel actin-containing nuclear complex. Actin binds directly to the hATAC subunit KAT14, and modulates its histone acetyl transferase activity in vitro and in cells. Transient interactions detected through BioID link actin to several steps of transcription as well as to RNA processing. Alterations in nuclear actin levels disturb alternative splicing in minigene assays, likely by affecting the transcription elongation rate. This interactome analysis thus identifies both novel direct binding partners and functional roles for nuclear actin, as well as forms a platform for further mechanistic studies on how actin operates during essential nuclear processes.This article has an associated First Person interview with the first author of the paper.

Proteomic and direct analysis in real time mass spectrometry analysis of a Native American ceremonial hat.

Complementary mass spectrometry analyses were performed to study a broken ceremonial hat of the Tlingit in the collection of the Smithsonian Institution National Museum of Natural History. The hat base and an associated cylinder are carved from wood and show multiple signs of age and breakage, as well as remnants of animal materials used for construction, decoration, and repair. Samples of animal tissues embedded in and attached to the wood were prepared for liquid chromatography-tandem mass spectrometry (LC-MS/MS), which identified proteins from five clades native to the object's area of origin. Surfaces on the hat and cylinder were analyzed using a direct analysis in real time (DART) MS system modified to accommodate the intact items. The presence of nicotine from tobacco smoke on the exterior and the relative absence of nicotine from the underside and formerly covered surfaces indicated that the cylinder was originally connected to the top of the hat. The characterization of the original object will be used to make informed decisions about reproduction of the intact hat for use by the Tlingit Kiks.ádi clan.

Candidate Treponema pallidum biomarkers uncovered in urine from individuals with syphilis using mass spectrometry.

A diagnostic test that could detect Treponema pallidum antigens in urine would facilitate the prompt diagnosis of syphilis. Urine from 54 individuals with various clinical stages of syphilis and 6 controls were pooled according to disease stage and interrogated with complementary mass spectrometry techniques to uncover potential syphilis biomarkers. In total, 26 unique peptides were uncovered corresponding to four unique T. pallidum proteins that have low genetic sequence similarity to other prokaryotes and human proteins. This is the first account of direct T. pallidum protein detection in human clinical samples using mass spectrometry. The implications of these findings for future diagnostic test development is discussed. Data are available via ProteomeXchange with identifier PXD009707.

Biomolecular Imaging of Regeneration of Zebrafish Caudal Fins Using High Spatial Resolution Ambient Mass Spectrometry.

We observed the molecular distribution changes that occurred during the regeneration of fresh zebrafish caudal fins using the recently developed ambient high-resolution mass spectrometry (MS) imaging technique of atmospheric pressure-nanoparticle and plasma-assisted laser desorption ionization (AP-nanoPALDI). AP-nanoPALDI analyses of fresh zebrafish caudal fins revealed that the small molecules, including neurotransmitters, amino acids, lipids, and metabolites of the regenerated area, were more evenly distributed throughout the bony rays and inter-ray mesenchymal tissues compared to the original area in the early stage. Zebrafish caudal fins of less than 200 μm thickness can be very useful for tissue regeneration studies using ambient MS imaging by providing sufficient biomolecular information at the molecular level for wound-healing studies. AP-nanoPALDI imaging was compared with a complementary MS imaging tool, surface sensitive time-of-flight secondary ion MS (ToF-SIMS).

Investigation of proteins in samples of a mid-18th century colonial mural painting by MALDI-TOF/MS and LC-ESI/MS (Orbitrap)

In this paper, we report the study of proteinaceous binders in samples from a South American Colonial 18th century painting using two complementary mass spectrometry techniques. Before restoration, seven micro-samples were extracted from representative colors of one of the mural paintings located inside the church of Our Lady of Copacabana de Andamarca built in 1723 in Bolivia. Previous analysis by gas chromatography (GC) of the amino acid derivatives of the extracted protein fraction of three of the samples, suggested the presence of animal glue and egg. In this work, we introduce a methodology that combines a protein extraction procedure with a typical treatment for analysis of proteins as tryptic peptides by mass spectrometry. For this study, we applied in the first stage, MALDI-TOF/MS and then, LC-ESI/MS (Orbitrap), an ultrahigh resolution mass spectrometry, to achieve more reliability in the identification of the protein binders.By LC-ESI/MS (Orbitrap), we detected several peptides from egg white proteins, particularly ovalbumin, ovotransferrin and lysozyme, and egg yolk proteins, vitellogenin-2 and apolipoprotein B, with high confidence peptides each. In all the samples, the presence of collagen from animal provenance was established by MALDI-TOF/MS and LC-ESI/MS (Orbitrap).To our knowledge, this is the first time that protein binders in Andean paintings are identified without ambiguity by MALDI-TOF/MS and LC-ESI/MS (Orbitrap) mass spectrometry using a proteomic approach.

MetaboRank: network-based recommendation system to interpret and enrich metabolomics results.

MotivationMetabolomics has shown great potential to improve the understanding of complex diseases, potentially leading to therapeutic target identification. However, no single analytical method allows monitoring all metabolites in a sample, resulting in incomplete metabolic fingerprints. This incompleteness constitutes a stumbling block to interpretation, raising the need for methods that can enrich those fingerprints. We propose MetaboRank, a new solution inspired by social network recommendation systems for the identification of metabolites potentially related to a metabolic fingerprint.ResultsMetaboRank method had been used to enrich metabolomics data obtained on cerebrospinal fluid samples from patients suffering from hepatic encephalopathy (HE). MetaboRank successfully recommended metabolites not present in the original fingerprint. The quality of recommendations was evaluated by using literature automatic search, in order to check that recommended metabolites could be related to the disease. Complementary mass spectrometry experiments and raw data analysis were performed to confirm these suggestions. In particular, MetaboRank recommended the overlooked α-ketoglutaramate as a metabolite which should be added to the metabolic fingerprint of HE, thus suggesting that metabolic fingerprints enhancement can provide new insight on complex diseases.Availability and implementationMethod is implemented in the MetExplore server and is available at www.metexplore.fr. A tutorial is available at https://metexplore.toulouse.inra.fr/com/tutorials/MetaboRank/2017-MetaboRank.pdf.Supplementary information Supplementary data are available at Bioinformatics online.

Metabolome Integrated Analysis of High-Temperature Response in Pinus radiata.

The integrative omics approach is crucial to identify the molecular mechanisms underlying high-temperature response in non-model species. Based on future scenarios of heat increase, Pinus radiata plants were exposed to a temperature of 40°C for a period of 5 days, including recovered plants (30 days after last exposure to 40°C) in the analysis. The analysis of the metabolome using complementary mass spectrometry techniques (GC-MS and LC-Orbitrap-MS) allowed the reliable quantification of 2,287 metabolites. The analysis of identified metabolites and highlighter metabolic pathways across heat time exposure reveal the dynamism of the metabolome in relation to high-temperature response in P. radiata, identifying the existence of a turning point (on day 3) at which P. radiata plants changed from an initial stress response program (shorter-term response) to an acclimation one (longer-term response). Furthermore, the integration of metabolome and physiological measurements, which cover from the photosynthetic state to hormonal profile, suggests a complex metabolic pathway interaction network related to heat-stress response. Cytokinins (CKs), fatty acid metabolism and flavonoid and terpenoid biosynthesis were revealed as the most important pathways involved in heat-stress response in P. radiata, with zeatin riboside (ZR) and isopentenyl adenosine (iPA) as the key hormones coordinating these multiple and complex interactions. On the other hand, the integrative approach allowed elucidation of crucial metabolic mechanisms involved in heat response in P. radiata, as well as the identification of thermotolerance metabolic biomarkers (L-phenylalanine, hexadecanoic acid, and dihydromyricetin), crucial metabolites which can reschedule the metabolic strategy to adapt to high temperature.

The Drosophila CLAMP protein associates with diverse proteins on chromatin.

Gaining new insights into gene regulation involves an in-depth understanding of protein-protein interactions on chromatin. A powerful model for studying mechanisms of gene regulation is dosage compensation, a process that targets the X-chromosome to equalize gene expression between XY males and XX females. We previously identified a zinc finger protein in Drosophila melanogaster that plays a sex-specific role in targeting the Male-specific lethal (MSL) dosage compensation complex to the male X-chromosome, called the Chromatin-Linked Adapter for MSL Proteins (CLAMP). More recently, we established that CLAMP has non-sex-specific roles as an essential protein that regulates chromatin accessibility at promoters genome-wide. To identify associations between CLAMP and other factors in both male and female cells, we used two complementary mass spectrometry approaches. We demonstrate that CLAMP associates with the transcriptional regulator complex Negative Elongation Factor (NELF) in both sexes and determine that CLAMP reduces NELF recruitment to several target genes. In sum, we have identified many new CLAMP-associated factors and provide a resource for further study of this little understood essential protein.

System-wide analysis of short-term response to high temperature in Pinus radiata.

Pinus radiata seedlings, the most widely planted pine species in the world, were exposed to temperatures within a range mimicking future scenarios based on current models of heat increase. The short-term heat response in P. radiata was studied in detail by exploring the metabolome, proteome and targeted transcriptome. The use of complementary mass spectrometry techniques, GC-MS and LC-Orbitrap-MS, together with novel bioinformatics tools allowed the reliable quantification of 2,075 metabolites and 901 protein groups. Integrative analyses of different functional levels and plant physiological status revealed a complex molecular interaction network of positive and negative correlations between proteins and metabolites involved in short-term heat response, including three main physiological functions as: 1) A hormone subnetwork, where fatty acids, flavonoids and hormones presented a key role; 2) An oxidoreductase subnetwork, including several dehydrogenase and peroxidase proteins; and 3) A heat shock protein subnetwork, with numerous proteins that contain a HSP20 domain, all of which were overexpressed at the transcriptional level. Integrated analysis pinpointed the basic mechanisms underlying the short-term physiological reaction of P. radiata during heat response. This approach was feasible in forest species and unmasked two novel candidate biomarkers of heat resistance, PHO1 and TRANSCRIPTION FACTOR APFI, and a MITOCHONDRIAL SMALL HEAT SHOCK PROTEIN, for use in future breeding programs.

Characterizing the Syphilis-Causing Treponema pallidum ssp. pallidum Proteome Using Complementary Mass Spectrometry.

BackgroundThe spirochete bacterium Treponema pallidum ssp. pallidum is the etiological agent of syphilis, a chronic multistage disease. Little is known about the global T. pallidum proteome, therefore mass spectrometry studies are needed to bring insights into pathogenicity and protein expression profiles during infection.Methodology/Principal FindingsTo better understand the T. pallidum proteome profile during infection, we studied T. pallidum ssp. pallidum DAL-1 strain bacteria isolated from rabbits using complementary mass spectrometry techniques, including multidimensional peptide separation and protein identification via matrix-assisted laser desorption ionization-time of flight (MALDI-TOF/TOF) and electrospray ionization (ESI-LTQ-Orbitrap) tandem mass spectrometry. A total of 6033 peptides were detected, corresponding to 557 unique T. pallidum proteins at a high level of confidence, representing 54% of the predicted proteome. A previous gel-based T. pallidum MS proteome study detected 58 of these proteins. One hundred fourteen of the detected proteins were previously annotated as hypothetical or uncharacterized proteins; this is the first account of 106 of these proteins at the protein level. Detected proteins were characterized according to their predicted biological function and localization; half were allocated into a wide range of functional categories. Proteins annotated as potential membrane proteins and proteins with unclear functional annotations were subjected to an additional bioinformatics pipeline analysis to facilitate further characterization. A total of 116 potential membrane proteins were identified, of which 16 have evidence supporting outer membrane localization. We found 8/12 proteins related to the paralogous tpr gene family: TprB, TprC/D, TprE, TprG, TprH, TprI and TprJ. Protein abundance was semi-quantified using label-free spectral counting methods. A low correlation (r = 0.26) was found between previous microarray signal data and protein abundance.ConclusionsThis is the most comprehensive description of the global T. pallidum proteome to date. These data provide valuable insights into in vivo T. pallidum protein expression, paving the way for improved understanding of the pathogenicity of this enigmatic organism.

Identification of Novel N-Glycosylation Sites at Noncanonical Protein Consensus Motifs.

N-glycosylation of proteins is well known to occur at asparagine residues that fall within the canonical consensus sequence N-X-S/T but has also been identified at a small number of asparagine residues within N-X-C motifs, including the N491 residue of human serotransferrin. Here we report novel glycosylation sites within noncanonical consensus motifs, in the conformation N-X-C, based on mass spectrometry analysis of partially deglycosylated glycopeptide targets. Alpha-1-acid glycoprotein (A1AG) and serotransferrin (Tf) were observed for the first time to be N-glycosylated on asparagine residues within a total of six unique noncanonical motifs. N-glycosylation was initially predicted in silico based on the evolutionary conservation of the N-X-C motif among related mammalian species and demonstrated experimentally in A1AG from porcine, canine, and feline sources and in human serotransferrin. High-resolution liquid chromatography-tandem mass spectrometry was employed to collect fragmentation data of predicted GlcNAcylated peptides and to assign modification sites within N-X-C motifs. A combination of targeted analytical techniques that includes complementary mass spectrometry platforms, enzymatic digestions, and partial-deglycosylation procedures was developed to confirm the novel observations. Additionally, we found that A1AG in porcine and canine sources is highly N-glycosylated at a noncanonical motif (N-Q-C) based on semiquantitative multiple reaction monitoring analysis-the first report of an N-X-C motif exhibiting substantial N-glycosylation. Although reports of N-X-C motif N-glycosylation are relatively uncommon in the literature, this work adds to a growing list of glycoproteins reported with glycosylation at various forms of noncanonical motifs.

Deciphering the Interplay among Multisite Phosphorylation, Interaction Dynamics, and Conformational Transitions in a Tripartite Protein System.

Multisite phosphorylation is a common pathway to regulate protein function, activity, and interaction pattern in vivo, but routine biochemical analysis is often insufficient to identify the number and order of individual phosphorylation reactions and their mechanistic impact on the protein behavior. Here, we integrate complementary mass spectrometry (MS)-based approaches to characterize a multisite phosphorylation-regulated protein system comprising Polo-like kinase 1 (Plk1) and its coactivators Aurora kinase A (Aur-A) and Bora, the interplay of which is essential for mitotic entry after DNA damage-induced cell cycle arrest. Native MS and cross-linking–MS revealed that Aur-A/Bora-mediated Plk1 activation is accompanied by the formation of Aur-A/Bora and Plk1/Bora heterodimers. We found that the Aur-A/Bora interaction is independent of the Bora phosphorylation state, whereas the Plk1/Bora interaction is dependent on extensive Bora multisite phosphorylation. Bottom-up and top-down proteomics analyses showed that Bora multisite phosphorylation proceeds via a well-ordered sequence of site-specific phosphorylation reactions, whereby we could reveal the involvement of up to 16 phosphorylated Bora residues. Ion mobility spectrometry–MS demonstrated that this multisite phosphorylation primes a substantial structural rearrangement of Bora, explaining the interdependence between extensive Bora multisite phosphorylation and Plk1/Bora complex formation. These results represent a first benchmark of our multipronged MS strategy, highlighting its potential to elucidate the mechanistic and structural implications of multisite protein phosphorylation.

PGlyco: a pipeline for the identification of intact N-glycopeptides by using HCD- and CID-MS/MS and MS3.

Confident characterization of the microheterogeneity of protein glycosylation through identification of intact glycopeptides remains one of the toughest analytical challenges for glycoproteomics. Recently proposed mass spectrometry (MS)-based methods still have some defects such as lack of the false discovery rate (FDR) analysis for the glycan identification and lack of sufficient fragmentation information for the peptide identification. Here we proposed pGlyco, a novel pipeline for the identification of intact glycopeptides by using complementary MS techniques: 1) HCD-MS/MS followed by product-dependent CID-MS/MS was used to provide complementary fragments to identify the glycans, and a novel target-decoy method was developed to estimate the false discovery rate of the glycan identification; 2) data-dependent acquisition of MS3 for some most intense peaks of HCD-MS/MS was used to provide fragments to identify the peptide backbones. By integrating HCD-MS/MS, CID-MS/MS and MS3, intact glycopeptides could be confidently identified. With pGlyco, a standard glycoprotein mixture was analyzed in the Orbitrap Fusion, and 309 non-redundant intact glycopeptides were identified with detailed spectral information of both glycans and peptides.