DOT1L regulates chromatin reorganization and gene expression during sperm differentiation.

Spermatozoa have a unique genome organization. Their chromatin is almost completely devoid of histones and is formed instead of protamines, which confer a high level of compaction and preserve paternal genome integrity until fertilization. Histone-to-protamine transition takes place in spermatids and is indispensable for the production of functional sperm. Here, we show that the H3K79-methyltransferase DOT1L controls spermatid chromatin remodeling and subsequent reorganization and compaction of the spermatozoon genome. Using a mouse model in which Dot1l is knocked-out (KO) in postnatal male germ cells, we found that Dot1l-KO sperm chromatin is less compact and has an abnormal content, characterized by the presence of transition proteins, immature protamine 2 forms and a higher level of histones. Proteomic and transcriptomic analyses performed on spermatids reveal that Dot1l-KO modifies the chromatin prior to histone removal and leads to the deregulation of genes involved in flagellum formation and apoptosis during spermatid differentiation. As a consequence of these chromatin and gene expression defects, Dot1l-KO spermatozoa have less compact heads and are less motile, which results in impaired fertility.

CK2ß Is a Gatekeeper of Focal Adhesions Regulating Cell Spreading.

CK2 is a hetero-tetrameric serine/threonine protein kinase made up of two CK2α/α' catalytic subunits and two CK2ß regulatory subunits. The free CK2α subunit and the tetrameric holoenzyme have distinct substrate specificity profiles, suggesting that the spatiotemporal organization of the individual CK2 subunits observed in living cells is crucial in the control of the many cellular processes that are governed by this pleiotropic kinase. Indeed, previous studies reported that the unbalanced expression of CK2 subunits is sufficient to drive epithelial to mesenchymal transition (EMT), a process involved in cancer invasion and metastasis. Moreover, sub-stoichiometric expression of CK2ß compared to CK2α in a subset of breast cancer tumors was correlated with the induction of EMT markers and increased epithelial cell plasticity in breast carcinoma progression. Phenotypic changes of epithelial cells are often associated with the activation of phosphotyrosine signaling. Herein, using phosphotyrosine enrichment coupled with affinity capture and proteomic analysis, we show that decreased expression of CK2ß in MCF10A mammary epithelial cells triggers the phosphorylation of a number of proteins on tyrosine residues and promotes the striking activation of the FAK1-Src-PAX1 signaling pathway. Moreover, morphometric analyses also reveal that CK2ß loss increases the number and the spatial distribution of focal adhesion signaling complexes that coordinate the adhesive and migratory processes. Together, our findings allow positioning CK2ß as a gatekeeper for cell spreading by restraining focal adhesion formation and invasion of mammary epithelial cells.

Analysis of the Arabidopsis coilin mutant reveals a positive role of AtCOILIN in plant immunity.

Biogenesis of ribonucleoproteins occurs in dynamic subnuclear compartments called Cajal bodies (CBs). COILIN is a critical scaffolding component essential for CB formation, composition, and activity. We recently showed that Arabidopsis (Arabidopsis thaliana) AtCOILIN is phosphorylated in response to bacterial elicitor treatment. Here, we further investigated the role of AtCOILIN in plant innate immunity. Atcoilin mutants are compromised in defense responses to bacterial pathogens. Besides confirming a role of AtCOILIN in alternative splicing (AS), Atcoilin showed differential expression of genes that are distinct from those of AS, including factors involved in RNA biogenesis, metabolism, plant immunity, and phytohormones. Atcoilin mutant plants have reduced levels of defense phytohormones. As expected, the mutant plants were more sensitive to the necrotrophic fungal pathogen Botrytis cinerea. Our findings reveal an important role for AtCOILIN in innate plant immunity.

Small Mass but Strong Information: Diagnostic Ions Provide Crucial Clues to Correctly Identify Histone Lysine Modifications.

(1) Background: The proteomic analysis of histones constitutes a delicate task due to the combination of two factors: slight variations in the amino acid sequences of variants and the multiplicity of post-translational modifications (PTMs), particularly those occurring on lysine residues. (2) Methods: To dissect the relationship between both aspects, we carefully evaluated PTM identification on lysine 27 from histone H3 (H3K27) and the artefactual chemical modifications that may lead to erroneous PTM determination. H3K27 is a particularly interesting example because it can bear a range of PTMs and it sits nearby residues 29 and 31 that vary between H3 sequence variants. We discuss how the retention times, neutral losses and immonium/diagnostic ions observed in the MS/MS spectra of peptides bearing modified lysines detectable in the low-mass region might help validate the identification of modified sequences. (3) Results: Diagnostic ions carry key information, thereby avoiding potential mis-identifications due to either isobaric PTM combinations or isobaric amino acid-PTM combinations. This also includes cases where chemical formylation or acetylation of peptide N-termini artefactually occurs during sample processing or simply in the timeframe of LC-MS/MS analysis. Finally, in the very subtle case of positional isomers possibly corresponding to a given mass of lysine modification, the immonium and diagnostic ions may allow the identification of the in vivo structure.

Chromatin phosphoproteomics unravels a function for AT-hook motif nuclear localized protein AHL13 in PAMP-triggered immunity.

In many eukaryotic systems during immune responses, mitogen-activated protein kinases (MAPKs) link cytoplasmic signaling to chromatin events by targeting transcription factors, chromatin remodeling complexes, and the RNA polymerase machinery. So far, knowledge on these events is scarce in plants and no attempts have been made to focus on phosphorylation events of chromatin-associated proteins. Here we carried out chromatin phosphoproteomics upon elicitor-induced activation of Arabidopsis The events in WT were compared with those in mpk3, mpk4, and mpk6 mutant plants to decipher specific MAPK targets. Our study highlights distinct signaling networks involving MPK3, MPK4, and MPK6 in chromatin organization and modification, as well as in RNA transcription and processing. Among the chromatin targets, we characterized the AT-hook motif containing nuclear localized (AHL) DNA-binding protein AHL13 as a substrate of immune MAPKs. AHL13 knockout mutant plants are compromised in pathogen-associated molecular pattern (PAMP)-induced reactive oxygen species production, expression of defense genes, and PAMP-triggered immunity. Transcriptome analysis revealed that AHL13 regulates key factors of jasmonic acid biosynthesis and signaling and affects immunity toward Pseudomonas syringae and Botrytis cinerea pathogens. Mutational analysis of the phosphorylation sites of AHL13 demonstrated that phosphorylation regulates AHL13 protein stability and thereby its immune functions.

Systematic genetic and proteomic screens during gametogenesis identify H2BK34 methylation as an evolutionary conserved meiotic mark.

BACKGROUND: Gametes are highly differentiated cells specialized to carry and protect the parental genetic information. During male germ cell maturation, histone proteins undergo distinct changes that result in a highly compacted chromatin organization. Technical difficulties exclude comprehensive analysis of precise histone mutations during mammalian spermatogenesis. The model organism Saccharomyces cerevisiae possesses a differentiation pathway termed sporulation which exhibits striking similarities to mammalian spermatogenesis. This study took advantage of this yeast pathway to first perform systematic mutational and proteomics screens on histones, revealing amino acid residues which are essential for the formation of spores. METHODS: A systematic mutational screen has been performed on the histones H2A and H2B, generating ~ 250 mutants using two genetic backgrounds and assessing their ability to form spores. In addition, histones were purified at key stages of sporulation and post-translational modifications analyzed by mass spectrometry. RESULTS: The mutation of 75 H2A H2B residues affected sporulation, many of which were localized to the nucleosome lateral surface. The use of different genetic backgrounds confirmed the importance of many of the residues, as 48% of yeast histone mutants exhibited impaired formation of spores in both genetic backgrounds. Extensive proteomic analysis identified 67 unique post-translational modifications during sporulation, 27 of which were previously unreported in yeast. Furthermore, 33 modifications are located on residues that were found to be essential for efficient sporulation in our genetic mutation screens. The quantitative analysis of these modifications revealed a massive deacetylation of all core histones during the pre-meiotic phase and a close interplay between H4 acetylation and methylation during yeast sporulation. Methylation of H2BK37 was also identified as a new histone marker of meiosis and the mouse paralog, H2BK34, was also enriched for methylation during meiosis in the testes, establishing conservation during mammalian spermatogenesis. CONCLUSION: Our results demonstrate that a combination of genetic and proteomic approaches applied to yeast sporulation can reveal new aspects of chromatin signaling pathways during mammalian spermatogenesis.

Multi-omic analysis of gametogenesis reveals a novel signature at the promoters and distal enhancers of active genes.

Epigenetic regulation of gene expression is tightly controlled by the dynamic modification of histones by chemical groups, the diversity of which has largely expanded over the past decade with the discovery of lysine acylations, catalyzed from acyl-coenzymes A. We investigated the dynamics of lysine acetylation and crotonylation on histones H3 and H4 during mouse spermatogenesis. Lysine crotonylation appeared to be of significant abundance compared to acetylation, particularly on Lys27 of histone H3 (H3K27cr) that accumulates in sperm in a cleaved form of H3. We identified the genomic localization of H3K27cr and studied its effects on transcription compared to the classical active mark H3K27ac at promoters and distal enhancers. The presence of both marks was strongly associated with highest gene expression. Assessment of their co-localization with transcription regulators (SLY, SOX30) and chromatin-binding proteins (BRD4, BRDT, BORIS and CTCF) indicated systematic highest binding when both active marks were present and different selective binding when present alone at chromatin. H3K27cr and H3K27ac finally mark the building of some sperm super-enhancers. This integrated analysis of omics data provides an unprecedented level of understanding of gene expression regulation by H3K27cr in comparison to H3K27ac, and reveals both synergistic and specific actions of each histone modification.

The Arabidopsis homolog of human G3BP1 is a key regulator of stomatal and apoplastic immunity.

Mammalian Ras-GTPase-activating protein SH3-domain-binding proteins (G3BPs) are a highly conserved family of RNA-binding proteins that link kinase receptor-mediated signaling to RNA metabolism. Mammalian G3BP1 is a multifunctional protein that functions in viral immunity. Here, we show that the Arabidopsis thaliana homolog of human G3BP1 negatively regulates plant immunity. Arabidopsis g3bp1 mutants showed enhanced resistance to the virulent bacterial pathogen Pseudomonas syringae pv. tomato. Pathogen resistance was mediated in Atg3bp1 mutants by altered stomatal and apoplastic immunity. Atg3bp1 mutants restricted pathogen entry into stomates showing insensitivity to bacterial coronatine-mediated stomatal reopening. AtG3BP1 was identified as a negative regulator of defense responses, which correlated with moderate up-regulation of salicylic acid biosynthesis and signaling without growth penalty.

The Trihelix transcription factor GT2-like 1 (GTL1) promotes salicylic acid metabolism, and regulates bacterial-triggered immunity.

The Trihelix Transcription factor GT2-like 1 (GTL1) was previously shown to be a key regulator of ploidy-dependent trichome growth and drought tolerance. Here, we report that GTL1 plays an important role in coordinating plant immunity. We show that gtl1 mutants are compromised in the regulation of basal immunity, microbial pattern-triggered immunity (PTI) and effector-triggered RIN4-mediated immunity. Transcriptome analysis revealed that GTL1 positively regulates defense genes and inhibits factors that mediate growth and development. By performing hormonal measurements and chromatin-immunoprecipitation studies, we found GTL1 to coordinate genes involved in salicylic acid metabolism, transport and response. Interaction studies and comparative transcriptomics to known data sets revealed that GTL1 is part of the MPK4 pathway and regulates oppositely the expression of differentially expressed genes in mpk4 plants. We introduced the gtl1 mutation in the mpk4 mutant and thereby partially suppressed its dwarfism and the high resistance against a bacterial invader. Our data show that GTL1 is part of the MPK4 pathway and acts as a positive regulator of bacterial-triggered immunity and SA homeostasis.

Proteomic Analysis of Histone Variants and Their PTMs: Strategies and Pitfalls.

Epigenetic modifications contribute to the determination of cell fate and differentiation. The molecular mechanisms underlying histone variants and post-translational modifications (PTMs) have been studied in the contexts of development, differentiation, and disease. Antibody-based assays have classically been used to target PTMs, but these approaches fail to reveal combinatorial patterns of modifications. In addition, some histone variants are so similar to canonical histones that antibodies have difficulty distinguishing between these isoforms. Mass spectrometry (MS) has progressively developed as a powerful technology for the study of histone variants and their PTMs. Indeed, MS analyses highlighted exquisitely complex combinations of PTMs, suggesting “crosstalk” between them, and also revealed that PTM patterns are often variant-specific. Even though the sensitivity and acquisition speed of MS instruments have considerably increased alongside the development of computational tools for the study of multiple PTMs, it remains challenging to correctly describe the landscape of histone PTMs, and in particular to confidently assign modifications to specific amino acids. Here, we provide an inventory of MS-based strategies and of the pitfalls inherent to histone PTM and variant characterization, while stressing the complex interplay between PTMs and histone sequence variations. We will particularly illustrate the roles played by MS-based analyses in identifying and quantifying histone variants and modifications.

Systematic quantitative analysis of H2A and H2B variants by targeted proteomics.

BACKGROUND: Histones organize DNA into chromatin through a variety of processes. Among them, a vast diversity of histone variants can be incorporated into chromatin and finely modulate its organization and functionality. Classically, the study of histone variants has largely relied on antibody-based assays. However, antibodies have a limited efficiency to discriminate between highly similar histone variants. RESULTS: In this study, we established a mass spectrometry-based analysis to address this challenge. We developed a targeted proteomics method, using selected reaction monitoring or parallel reaction monitoring, to quantify a maximum number of histone variants in a single multiplexed assay, even when histones are present in a crude extract. This strategy was developed on H2A and H2B variants, using 55 peptides corresponding to 25 different histone sequences, among which a few differ by a single amino acid. The methodology was then applied to mouse testis extracts in which almost all histone variants are expressed. It confirmed the abundance profiles of several testis-specific histones during successive stages of spermatogenesis and the existence of predicted H2A.L.1 isoforms. This methodology was also used to explore the over-expression pattern of H2A.L.1 isoforms in a mouse model of male infertility. CONCLUSIONS: Our results demonstrate that targeted proteomics is a powerful method to quantify highly similar histone variants and isoforms. The developed method can be easily transposed to the study of human histone variants, whose abundance can be deregulated in various diseases.

Quantitative Phosphoproteomic Analysis Reveals Shared and Specific Targets of Arabidopsis Mitogen-Activated Protein Kinases (MAPKs) MPK3, MPK4, and MPK6.

In Arabidopsis, mitogen-activated protein kinases MPK3, MPK4, and MPK6 constitute essential relays for a variety of functions including cell division, development and innate immunity. Although some substrates of MPK3, MPK4 and MPK6 have been identified, the picture is still far from complete. To identify substrates of these MAPKs likely involved in cell division, growth and development we compared the phosphoproteomes of wild-type and mpk3, mpk4, and mpk6. To study the function of these MAPKs in innate immunity, we analyzed their phosphoproteomes following microbe-associated molecular pattern (MAMP) treatment. Partially overlapping substrates were retrieved for all three MAPKs, showing target specificity to one, two or all three MAPKs in different biological processes. More precisely, our results illustrate the fact that the entity to be defined as a specific or a shared substrate for MAPKs is not a phosphoprotein but a particular (S/T)P phosphorylation site in a given protein. One hundred fifty-two peptides were identified to be differentially phosphorylated in response to MAMP treatment and/or when compared between genotypes and 70 of them could be classified as putative MAPK targets. Biochemical analysis of a number of putative MAPK substrates by phosphorylation and interaction assays confirmed the global phosphoproteome approach. Our study also expands the set of MAPK substrates to involve other protein kinases, including calcium-dependent (CDPK) and sugar nonfermenting (SnRK) protein kinases.

Hepatitis C virus core protein targets 4E-BP1 expression and phosphorylation and potentiates Myc-induced liver carcinogenesis in transgenic mice.

Hepatitis C virus (HCV) is a leading cause of liver diseases including the development of hepatocellular carcinoma (HCC). Particularly, core protein has been involved in HCV-related liver pathologies. However, the impact of HCV core on signaling pathways supporting the genesis of HCC remains largely elusive. To decipher the host cell signaling pathways involved in the oncogenic potential of HCV core, a global quantitative phosphoproteomic approach was carried out. This study shed light on novel differentially phosphorylated proteins, in particular several components involved in translation. Among the eukaryotic initiation factors that govern the translational machinery, 4E-BP1 represents a master regulator of protein synthesis that is associated with the development and progression of cancers due to its ability to increase protein expression of oncogenic pathways. Enhanced levels of 4E-BP1 in non-modified and phosphorylated forms were validated in human hepatoma cells and in mouse primary hepatocytes expressing HCV core, in the livers of HCV core transgenic mice as well as in HCV-infected human primary hepatocytes. The contribution of HCV core in carcinogenesis and the status of 4E-BP1 expression and phosphorylation were studied in HCV core/Myc double transgenic mice. HCV core increased the levels of 4E-BP1 expression and phosphorylation and significantly accelerated the onset of Myc-induced tumorigenesis in these double transgenic mice. These results reveal a novel function of HCV core in liver carcinogenesis potentiation. They position 4E-BP1 as a tumor-specific target of HCV core and support the involvement of the 4E-BP1/eIF4E axis in hepatocarcinogenesis.

MS_HistoneDB, a manually curated resource for proteomic analysis of human and mouse histones.

BACKGROUND: Histones and histone variants are essential components of the nuclear chromatin. While mass spectrometry has opened a large window to their characterization and functional studies, their identification from proteomic data remains challenging. Indeed, the current interpretation of mass spectrometry data relies on public databases which are either not exhaustive (Swiss-Prot) or contain many redundant entries (UniProtKB or NCBI). Currently, no protein database is ideally suited for the analysis of histones and the complex array of mammalian histone variants. RESULTS: We propose two proteomics-oriented manually curated databases for mouse and human histone variants. We manually curated >1700 gene, transcript and protein entries to produce a non-redundant list of 83 mouse and 85 human histones. These entries were annotated in accordance with the current nomenclature and unified with the "HistoneDB2.0 with Variants" database. This resource is provided in a format that can be directly read by programs used for mass spectrometry data interpretation. In addition, it was used to interpret mass spectrometry data acquired on histones extracted from mouse testis. Several histone variants, which had so far only been inferred by homology or detected at the RNA level, were detected by mass spectrometry, confirming the existence of their protein form. CONCLUSIONS: Mouse and human histone entries were collected from different databases and subsequently curated to produce a non-redundant protein-centric resource, MS_HistoneDB. It is dedicated to the proteomic study of histones in mouse and human and will hopefully facilitate the identification and functional study of histone variants.

Protein complexes characterization in Arabidopsis thaliana by tandem affinity purification coupled to mass spectrometry analysis.

Proteins are major elements participating in all the key functions of the cells. They rarely fulfill their physiological roles in an autonomous way but rather act as part of more complex cellular machines. Indeed they can bind different types of molecules (proteins, nucleic acids, metabolites, etc.), via stable or transient interactions, depending on their nature and functions. The identification of the molecular partners of a given protein is hence essential to better understand its roles, regulation, and mechanisms of action.This chapter describes the use of a tandem affinity purification approach followed by mass spectrometry analysis to try to identify and characterize the proteins involved in protein complexes in Arabidopsis thaliana and decipher some mechanisms of regulation of the modules. Important elements to consider in such an approach are first extensively exposed in the introduction. This technique, in combination with complementary approaches like yeast two-hybrid and bimolecular fluorescence complementation, can be an interesting source of data to identify and characterize in vivo protein complexes.

Phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins.

In eukaryotes, most of the DNA is located in the nucleus where it is organized with histone proteins in a higher order structure as chromatin. Chromatin and chromatin-associated proteins contribute to DNA-related processes such as replication and transcription as well as epigenetic regulation. Protein functions are often regulated by PTMs among which phosphorylation is one of the most abundant PTM. Phosphorylation of proteins affects important properties, such as enzyme activity, protein stability, or subcellular localization. We here describe the main specificities of protein phosphorylation in plants and review the current knowledge on phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins. We also outline some future challenges to further elucidate protein phosphorylation and chromatin regulation.

Proteomic and phosphoproteomic analyses of chromatin-associated proteins from Arabidopsis thaliana.

The nucleus is the organelle where basically all DNA-related processes take place in eukaryotes, such as replication, transcription, and splicing as well as epigenetic regulation. The identification and description of the nuclear proteins is one of the requisites toward a comprehensive understanding of the biological functions accomplished in the nucleus. Many of the regulatory mechanisms of protein functions rely on their PTMs among which phosphorylation is probably one of the most important properties affecting enzymatic activity, interaction with other molecules, localization, or stability. So far, the nuclear and subnuclear proteome and phosphoproteome of the model plant Arabidopsis thaliana have been the subject of very few studies. In this work, we developed a purification protocol of Arabidopsis chromatin-associated proteins and performed proteomic and phosphoproteomic analyses identifying a total of 879 proteins of which 198 were phosphoproteins that were mainly involved in chromatin remodeling, transcriptional regulation, and RNA processing. From 230 precisely localized phosphorylation sites (phosphosites), 52 correspond to hitherto unidentified sites. This protocol and data thereby obtained should be a valuable resource for many domains of plant research.

Identification of novel PAMP-triggered phosphorylation and dephosphorylation events in Arabidopsis thaliana by quantitative phosphoproteomic analysis.

Signaling cascades rely strongly on protein kinase-mediated substrate phosphorylation. Currently a major challenge in signal transduction research is to obtain high confidence substrate phosphorylation sites and assign them to specific kinases. In response to bacterial flagellin, a pathogen-associated molecular pattern (PAMP), we searched for rapidly phosphorylated proteins in Arabidopsis thaliana by combining multistage activation (MSA) and electron transfer dissociation (ETD) fragmentation modes, which generate complementary spectra and identify phosphopeptide sites with increased reliability. Of a total of 825 phosphopeptides, we identified 58 to be differentially phosphorylated. These peptides harbor kinase motifs of mitogen-activated protein kinases (MAPKs) and calcium-dependent protein kinases (CDPKs), as well as yet unknown protein kinases. Importantly, 12 of the phosphopeptides show reduced phosphorylation upon flagellin treatment. Since protein abundance levels did not change, these results indicate that flagellin induces not only various protein kinases but also protein phosphatases, even though a scenario of inhibited kinase activity may also be possible.

Brassinosteroid-regulated GSK3/Shaggy-like kinases phosphorylate mitogen-activated protein (MAP) kinase kinases, which control stomata development in Arabidopsis thaliana.

Brassinosteroids (BRs) are steroid hormones that coordinate fundamental developmental programs in plants. In this study we show that in addition to the well established roles of BRs in regulating cell elongation and cell division events, BRs also govern cell fate decisions during stomata development in Arabidopsis thaliana. In wild-type A. thaliana, stomatal distribution follows the one-cell spacing rule; that is, adjacent stomata are spaced by at least one intervening pavement cell. This rule is interrupted in BR-deficient and BR signaling-deficient A. thaliana mutants, resulting in clustered stomata. We demonstrate that BIN2 and its homologues, GSK3/Shaggy-like kinases involved in BR signaling, can phosphorylate the MAPK kinases MKK4 and MKK5, which are members of the MAPK module YODA-MKK4/5-MPK3/6 that controls stomata development and patterning. BIN2 phosphorylates a GSK3/Shaggy-like kinase recognition motif in MKK4, which reduces MKK4 activity against its substrate MPK6 in vitro. In vivo we show that MKK4 and MKK5 act downstream of BR signaling because their overexpression rescued stomata patterning defects in BR-deficient plants. A model is proposed in which GSK3-mediated phosphorylation of MKK4 and MKK5 enables for a dynamic integration of endogenous or environmental cues signaled by BRs into cell fate decisions governed by the YODA-MKK4/5-MPK3/6 module.

Automated phosphopeptide identification using multiple MS/MS fragmentation modes.

Phosphopeptide identification is still a challenging task because fragmentation spectra obtained by mass spectrometry do not necessarily contain sufficient fragment ions to establish with certainty the underlying amino acid sequence and the precise phosphosite. To improve upon this, it has been suggested to acquire pairs of spectra from every phosphorylated precursor ion using different fragmentation modes, for example CID, ETD, and/or HCD. The development of automated tools for the interpretation of these paired spectra has however, until now, lagged behind. Using phosphopeptide samples analyzed by an LTQ-Orbitrap instrument, we here assess an approach in which, on each selected precursor, a pair of CID spectra, with or without multistage activation (MSA or MS2, respectively), are acquired in the linear ion trap. We applied this approach on phosphopeptide samples of variable proteomic complexity obtained from Arabidopsis thaliana . We present a straightforward computational approach to reconcile sequence and phosphosite identifications provided by the database search engine Mascot on the spectrum pairs, using two simple filtering rules, at the amino acid sequence and phosphosite localization levels. If multiple sequences and/or phosphosites are likely, they are reported in the consensus sequence. Using our program FragMixer, we could assess that on samples of moderate complexity, it was worth combining the two fragmentation schemes on every precursor ion to help efficiently identify amino acid sequences and precisely localize phosphosites. FragMixer can be flexibly configured, independently of the Mascot search parameters, and can be applied to various spectrum pairs, such as MSA/ETD and ETD/HCD, to automatically compare and combine the information provided by these more differing fragmentation modes. The software is openly accessible and can be downloaded from our Web site at http://proteomics.fr/FragMixer.