Evolution of insect innate immunity through domestication of bacterial toxins.

Toxin cargo genes are often horizontally transferred by phages between bacterial species and are known to play an important role in the evolution of bacterial pathogenesis. Here, we show how these same genes have been horizontally transferred from phage or bacteria to animals and have resulted in novel adaptations. We discovered that two widespread bacterial genes encoding toxins of animal cells, cytolethal distending toxin subunit B (cdtB) and apoptosis-inducing protein of 56 kDa (aip56), were captured by insect genomes through horizontal gene transfer from bacteria or phages. To study the function of these genes in insects, we focused on Drosophila ananassae as a model. In the D. ananassae subgroup species, cdtB and aip56 are present as singular (cdtB) or fused copies (cdtB::aip56) on the second chromosome. We found that cdtB and aip56 genes and encoded proteins were expressed by immune cells, some proteins were localized to the wasp embryo's serosa, and their expression increased following parasitoid wasp infection. Species of the ananassae subgroup are highly resistant to parasitoid wasps, and we observed that D. ananassae lines carrying null mutations in cdtB and aip56 toxin genes were more susceptible to parasitoids than the wild type. We conclude that toxin cargo genes were captured by these insects millions of years ago and integrated as novel modules into their innate immune system. These modules now represent components of a heretofore undescribed defense response and are important for resistance to parasitoid wasps. Phage or bacterially derived eukaryotic toxin genes serve as macromutations that can spur the instantaneous evolution of novelty in animals.

Multiple Layers of Complexity in O-Glycosylation Illustrated With the Urinary Glycoproteome.

While N-glycopeptides are relatively easy to characterize, O-glycosylation analysis is more complex. In this article, we illustrate the multiple layers of O-glycopeptide characterization that make this task so challenging. We believe our carefully curated dataset represents perhaps the largest intact human glycopeptide mixture derived from individuals, not from cell lines. The samples were collected from healthy individuals, patients with superficial or advanced bladder cancer (three of each group), and a single bladder inflammation patient. The data were scrutinized manually and interpreted using three different search engines: Byonic, Protein Prospector, and O-Pair, and the tool MS-Filter. Despite all the recent advances, reliable automatic O-glycopeptide assignment has not been solved yet. Our data reveal such diversity of site-specific O-glycosylation that has not been presented before. In addition to the potential biological implications, this dataset should be a valuable resource for software developers in the same way as some of our previously released data has been used in the development of O-Pair and O-Glycoproteome Analyzer. Based on the manual evaluation of the performance of the existing tools with our data, we lined up a series of recommendations that if implemented could significantly improve the reliability of glycopeptide assignments.

Lectin-Based Immunophenotyping and Whole Proteomic Profiling of CT-26 Colon Carcinoma Murine Model.

A murine colorectal carcinoma (CRC) model was established. CT26 colon carcinoma cells were injected into BALB/c mice's spleen to study the primary tumor and the mechanisms of cell spread of colon cancer to the liver. The CRC was verified by the immunohistochemistry of Pan Cytokeratin and Vimentin expression. Immunophenotyping of leukocytes isolated from CRC-bearing BALB/c mice or healthy controls, such as CD19+ B cells, CD11+ myeloid cells, and CD3+ T cells, was carried out using fluorochrome-labeled lectins. The binding of six lectins to white blood cells, such as galectin-1 (Gal1), siglec-1 (Sig1), Sambucus nigra lectin (SNA), Aleuria aurantia lectin (AAL), Phytolacca americana lectin (PWM), and galectin-3 (Gal3), was assayed. Flow cytometric analysis of the splenocytes revealed the increased binding of SNA, and AAL to CD3 + T cells and CD11b myeloid cells; and increased siglec-1 and AAL binding to CD19 B cells of the tumor-bearing mice. The whole proteomic analysis of the established CRC-bearing liver and spleen versus healthy tissues identified differentially expressed proteins, characteristic of the primary or secondary CRC tissues. KEGG Gene Ontology bioinformatic analysis delineated the established murine CRC characteristic protein interaction networks, biological pathways, and cellular processes involved in CRC. Galectin-1 and S100A4 were identified as upregulated proteins in the primary and secondary CT26 tumor tissues, and these were previously reported to contribute to the poor prognosis of CRC patients. Modelling the development of liver colonization of CRC by the injection of CT26 cells into the spleen may facilitate the understanding of carcinogenesis in human CRC and contribute to the development of novel therapeutic strategies.

Proteotranscriptomic Discrimination of Tumor and Normal Tissues in Renal Cell Carcinoma.

Clear cell renal carcinoma is the most frequent type of kidney cancer, with an increasing incidence rate worldwide. In this research, we used a proteotranscriptomic approach to differentiate normal and tumor tissues in clear cell renal cell carcinoma (ccRCC). Using transcriptomic data of patients with malignant and paired normal tissue samples from gene array cohorts, we identified the top genes over-expressed in ccRCC. We collected surgically resected ccRCC specimens to further investigate the transcriptomic results on the proteome level. The differential protein abundance was evaluated using targeted mass spectrometry (MS). We assembled a database of 558 renal tissue samples from NCBI GEO and used these to uncover the top genes with higher expression in ccRCC. For protein level analysis 162 malignant and normal kidney tissue samples were acquired. The most consistently upregulated genes were IGFBP3, PLIN2, PLOD2, PFKP, VEGFA, and CCND1 (p < 10-5 for each gene). Mass spectrometry further validated the differential protein abundance of these genes (IGFBP3, p = 7.53 × 10-18; PLIN2, p = 3.9 × 10-39; PLOD2, p = 6.51 × 10-36; PFKP, p = 1.01 × 10-47; VEGFA, p = 1.40 × 10-22; CCND1, p = 1.04 × 10-24). We also identified those proteins which correlate with overall survival. Finally, a support vector machine-based classification algorithm using the protein-level data was set up. We used transcriptomic and proteomic data to identify a minimal panel of proteins highly specific for clear cell renal carcinoma tissues. The introduced gene panel could be used as a promising tool in the clinical setting.

Sperm-Leucylaminopeptidases are required for male fertility as structural components of mitochondrial paracrystalline material in Drosophila melanogaster sperm.

Drosophila melanogaster sperm reach an extraordinary long size, 1.8 mm, by the end of spermatogenesis. The mitochondrial derivatives run along the entire flagellum and provide structural rigidity for flagellar movement, but its precise function and organization is incompletely understood. The two mitochondrial derivatives differentiate and by the end of spermatogenesis the minor one reduces its size and the major one accumulates paracrystalline material inside it. The molecular constituents and precise function of the paracrystalline material have not yet been revealed. Here we purified the paracrystalline material from mature sperm and identified by mass spectrometry Sperm-Leucylaminopeptidase (S-Lap) family members as important constituents of it. To study the function of S-Lap proteins we show the characterization of classical mutants and RNAi lines affecting of the S-Lap genes and the analysis of their mutant phenotypes. We show that the male sterile phenotype of the S-Lap mutants is caused by defects in paracrystalline material accumulation and abnormal structure of the elongated major mitochondrial derivatives. Our work shows that S-Lap proteins localize and accumulate in the paracrystalline material of the major mitochondrial derivative. Therefore, we propose that S-Lap proteins are important constituents of the paracrystalline material of Drosophila melanogaster sperm.

STABILON, a Novel Sequence Motif That Enhances the Expression and Accumulation of Intracellular and Secreted Proteins.

The dynamic balance of transcriptional and translational regulation together with degron-controlled proteolysis shapes the ever-changing cellular proteome. While a large variety of degradation signals has been characterized, our knowledge of cis-acting protein motifs that can in vivo stabilize otherwise short-lived proteins is very limited. We have identified and characterized a conserved 13-mer protein segment derived from the p54/Rpn10 ubiquitin receptor subunit of the Drosophila 26S proteasome, which fulfills all the characteristics of a protein stabilization motif (STABILON). Attachment of STABILON to various intracellular as well as medically relevant secreted model proteins resulted in a significant increase in their cellular or extracellular concentration in mammalian cells. We demonstrate that STABILON acts as a universal and dual function motif that, on the one hand, increases the concentration of the corresponding mRNAs and, on the other hand, prevents the degradation of short-lived fusion proteins. Therefore, STABILON may lead to a breakthrough in biomedical recombinant protein production.

Arabidopsis RETINOBLASTOMA RELATED directly regulates DNA damage responses through functions beyond cell cycle control.

The rapidly proliferating cells in plant meristems must be protected from genome damage. Here, we show that the regulatory role of the Arabidopsis RETINOBLASTOMA RELATED (RBR) in cell proliferation can be separated from a novel function in safeguarding genome integrity. Upon DNA damage, RBR and its binding partner E2FA are recruited to heterochromatic γH2AX-labelled DNA damage foci in an ATM- and ATR-dependent manner. These γH2AX-labelled DNA lesions are more dispersedly occupied by the conserved repair protein, AtBRCA1, which can also co-localise with RBR foci. RBR and AtBRCA1 physically interact in vitro and in planta Genetic interaction between the RBR-silenced amiRBR and Atbrca1 mutants suggests that RBR and AtBRCA1 may function together in maintaining genome integrity. Together with E2FA, RBR is directly involved in the transcriptional DNA damage response as well as in the cell death pathway that is independent of SOG1, the plant functional analogue of p53. Thus, plant homologs and analogues of major mammalian tumour suppressor proteins form a regulatory network that coordinates cell proliferation with cell and genome integrity.

Microtubule organization in presynaptic boutons relies on the formin DAAM.

Regulation of the cytoskeleton is fundamental to the development and function of synaptic terminals, such as neuromuscular junctions. Despite the identification of numerous proteins that regulate synaptic actin and microtubule dynamics, the mechanisms of cytoskeletal control during terminal arbor formation have remained largely elusive. Here, we show that DAAM, a member of the formin family of cytoskeleton organizing factors, is an important presynaptic regulator of neuromuscular junction development in Drosophila We demonstrate that the actin filament assembly activity of DAAM plays a negligible role in terminal formation; rather, DAAM is necessary for synaptic microtubule organization. Genetic interaction studies consistently link DAAM with the Wg/Ank2/Futsch module of microtubule regulation and bouton formation. Finally, we provide evidence that DAAM is tightly associated with the synaptic active zone scaffold, and electrophysiological data point to a role in the modulation of synaptic vesicle release. Based on these results, we propose that DAAM is an important cytoskeletal effector element of the Wg/Ank2 pathway involved in the determination of basic synaptic structures, and, additionally, that DAAM may couple the active zone scaffold to the presynaptic cytoskeleton.

The interactome of the N-terminus of band 3 regulates red blood cell metabolism and storage quality.

Band 3 (anion exchanger 1; AE1) is the most abundant membrane protein in red blood cells, which in turn are the most abundant cells in the human body. A compelling model posits that, at high oxygen saturation, the N-terminal cytosolic domain of AE1 binds to and inhibits glycolytic enzymes, thus diverting metabolic fluxes to the pentose phosphate pathway to generate reducing equivalents. Dysfunction of this mechanism occurs during red blood cell aging or storage under blood bank conditions, suggesting a role for AE1 in the regulation of the quality of stored blood and efficacy of transfusion, a life-saving intervention for millions of recipients worldwide. Here we leveraged two murine models carrying genetic ablations of AE1 to provide mechanistic evidence of the role of this protein in the regulation of erythrocyte metabolism and storage quality. Metabolic observations in mice recapitulated those in a human subject lacking expression of AE11-11 (band 3 Neapolis), while common polymorphisms in the region coding for AE11-56 correlate with increased susceptibility to osmotic hemolysis in healthy blood donors. Through thermal proteome profiling and crosslinking proteomics, we provide a map of the red blood cell interactome, with a focus on AE11-56 and validate recombinant AE1 interactions with glyceraldehyde 3-phosphate dehydrogenase. As a proof-of-principle and to provide further mechanistic evidence of the role of AE1 in the regulation of redox homeo stasis of stored red blood cells, we show that incubation with a cell-penetrating AE11-56 peptide can rescue the metabolic defect in glutathione recycling and boost post-transfusion recovery of stored red blood cells from healthy human donors and genetically ablated mice.

Mass spectrometry-based molecular mapping of native FXIIIa cross-links in insoluble fibrin clots.

The roles of factor XIIIa-specific cross-links in thrombus formation, regression, or probability for embolization are largely unknown. A molecular understanding of fibrin architecture at the level of these cross-links could inform the development of therapeutic strategies to prevent the sequelae of thromboembolism. Here, we present an MS-based method to map native factor XIIIa cross-links in the insoluble matrix component of whole-blood or plasma-fibrin clots and in in vivo thrombi. Using a chaotrope-insoluble digestion method and quantitative cross-linking MS, we identified the previously mapped fibrinogen peptides that are responsible for covalent D-dimer association, as well as dozens of novel cross-links in the αC region of fibrinogen α. Our findings expand the known native cross-linked species from one to over 100 and suggest distinct antiparallel registries for interprotofibril association and covalent attachment of serpins that regulate clot dissolution.

Analysis of Mammalian O-Glycopeptides-We Have Made a Good Start, but There is a Long Way to Go.

Glycosylation is perhaps the most common post-translational modification. Recently there has been growing interest in cataloging the glycan structures, glycoproteins, and specific sites modified and deciphering the biological functions of glycosylation. Although the results are piling up for N-glycosylation, O-glycosylation is seriously trailing behind. In our review we reiterate the difficulties researchers have to overcome in order to characterize O-glycosylation. We describe how an ingenious cell engineering method delivered exciting results, and what could we gain from "wild-type" samples. Although we refer to the biological role(s) of O-glycosylation, we do not provide a complete inventory on this topic.

Extended Sialylated O-Glycan Repertoire of Human Urinary Glycoproteins Discovered and Characterized Using Electron-Transfer/Higher-Energy Collision Dissociation.

A relatively novel activation technique, electron-transfer/higher-energy collision dissociation (EThcD) was used in the LC-MS/MS analysis of tryptic glycopeptides enriched with wheat germ agglutinin from human urine samples. We focused on the characterization of mucin-type O-glycopeptides. EThcD in a single spectrum provided information on both the peptide modified and the glycan carried. Unexpectedly, glycan oxonium ions indicated the presence of O-acetyl, and even O-diacetyl-sialic acids. B and Y fragment ions revealed that (i) in core 1 structures the Gal residue featured the O-acetyl-sialic acid, when there was only one in the glycan; (ii) several glycopeptides featured core 1 glycans with disialic acids, in certain instances O-acetylated; (iii) the disialic acid was linked to the GalNAc residue whatever the degree of O-acetylation; (iv) core 2 isomers with a single O-acetyl-sialic acid were chromatographically resolved. Glycan fragmentation also helped to decipher additional core 2 oligosaccharides: a LacdiNAc-like structure, glycans carrying sialyl LewisX/A at different stages of O-acetylation, and blood antigens. A sialo core 3 structure was also identified. We believe this is the first study when such structures were characterized from a very complex mixture and were linked not only to a specific protein, but also the sites of modifications have been determined.

Transcriptional repression by MYB3R proteins regulates plant organ growth.

In multicellular organisms, temporal and spatial regulation of cell proliferation is central for generating organs with defined sizes and morphologies. For establishing and maintaining the post-mitotic quiescent state during cell differentiation, it is important to repress genes with mitotic functions. We found that three of the Arabidopsis MYB3R transcription factors synergistically maintain G2/M-specific genes repressed in post-mitotic cells and restrict the time window of mitotic gene expression in proliferating cells. The combined mutants of the three repressor-type MYB3R genes displayed long roots, enlarged leaves, embryos, and seeds. Genome-wide chromatin immunoprecipitation revealed that MYB3R3 binds to the promoters of G2/M-specific genes and to E2F target genes. MYB3R3 associates with the repressor-type E2F, E2FC, and the RETINOBLASTOMA RELATED proteins. In contrast, the activator MYB3R4 was in complex with E2FB in proliferating cells. With mass spectrometry and pairwise interaction assays, we identified some of the other conserved components of the multiprotein complexes, known as DREAM/dREAM in human and flies. In plants, these repressor complexes are important for periodic expression during cell cycle and to establish a post-mitotic quiescent state determining organ size.

Nectar- and stigma exudate-specific expression of an acidic chitinase could partially protect certain apple cultivars against fire blight disease.

MAIN CONCLUSION: Certain apple cultivars accumulate to high levels in their nectar and stigma exudate an acidic chitinase III protein that can protect against pathogens including fire blight disease causing Erwinia amylovora. To prevent microbial infections, flower nectars and stigma exudates contain various antimicrobial compounds. Erwinia amylovora, the causing bacterium of the devastating fire blight apple disease, is the model pathogen that multiplies in flower secretions and infects through the nectaries. Although Erwinia-resistant apples are not available, certain cultivars are tolerant. It was reported that in flower infection assay, the 'Freedom' cultivar was Erwinia tolerant, while the 'Jonagold' cultivar was susceptible. We hypothesized that differences in the nectar protein compositions lead to different susceptibility. Indeed, we found that an acidic chitinase III protein (Machi3-1) selectively accumulates to very high levels in the nectar and the stigma exudate of the 'Freedom' cultivar. We show that three different Machi3-1 alleles exist in apple cultivars and that only the 5B-Machi3-1 allele expresses the Machi3-1 protein in the nectar and the stigma exudate. We demonstrate that the 5B-Machi3-1 allele was introgressed from the Malus floribunda 821 clone into different apple cultivars including the 'Freedom'. Our data suggest that MYB-binding site containing repeats of the 5B-Machi3-1 promoter is responsible for the strong nectar- and stigma exudate-specific expression. As we found that in vitro, the Machi3-1 protein impairs growth and biofilm formation of Erwinia at physiological concentration, we propose that the Machi3-1 protein could partially protect 5B-Machi3-1 allele containing cultivars against Erwinia by inhibiting the multiplication and biofilm formation of the pathogen in the stigma exudate and in the nectar.

AtCRK5 Protein Kinase Exhibits a Regulatory Role in Hypocotyl Hook Development during Skotomorphogenesis.

Seedling establishment following germination requires the fine tuning of plant hormone levels including that of auxin. Directional movement of auxin has a central role in the associated processes, among others, in hypocotyl hook development. Regulated auxin transport is ensured by several transporters (PINs, AUX1, ABCB) and their tight cooperation. Here we describe the regulatory role of the Arabidopsis thaliana CRK5 protein kinase during hypocotyl hook formation/opening influencing auxin transport and the auxin-ethylene-GA hormonal crosstalk. It was found that the Atcrk5-1 mutant exhibits an impaired hypocotyl hook establishment phenotype resulting only in limited bending in the dark. The Atcrk5-1 mutant proved to be deficient in the maintenance of local auxin accumulation at the concave side of the hypocotyl hook as demonstrated by decreased fluorescence of the auxin sensor DR5::GFP. Abundance of the polar auxin transport (PAT) proteins PIN3, PIN7, and AUX1 were also decreased in the Atcrk5-1 hypocotyl hook. The AtCRK5 protein kinase was reported to regulate PIN2 protein activity by phosphorylation during the root gravitropic response. Here it is shown that AtCRK5 can also phosphorylate in vitro the hydrophilic loops of PIN3. We propose that AtCRK5 may regulate hypocotyl hook formation in Arabidopsis thaliana through the phosphorylation of polar auxin transport (PAT) proteins, the fine tuning of auxin transport, and consequently the coordination of auxin-ethylene-GA levels.

CRK5 Protein Kinase Contributes to the Progression of Embryogenesis of Arabidopsis thaliana.

The fine tuning of hormone (e.g., auxin and gibberellin) levels and hormone signaling is required for maintaining normal embryogenesis. Embryo polarity, for example, is ensured by the directional movement of auxin that is controlled by various types of auxin transporters. Here, we present pieces of evidence for the auxin-gibberellic acid (GA) hormonal crosstalk during embryo development and the regulatory role of the Arabidopsis thaliana Calcium-Dependent Protein Kinase-Related Kinase 5 (AtCRK5) in this regard. It is pointed out that the embryogenesis of the Atcrk5-1 mutant is delayed in comparison to the wild type. This delay is accompanied with a decrease in the levels of GA and auxin, as well as the abundance of the polar auxin transport (PAT) proteins PIN1, PIN4, and PIN7 in the mutant embryos. We have previously showed that AtCRK5 can regulate the PIN2 and PIN3 proteins either directly by phosphorylation or indirectly affecting the GA level during the root gravitropic and hypocotyl hook bending responses. In this manuscript, we provide evidence that the AtCRK5 protein kinase can in vitro phosphorylate the hydrophilic loops of additional PIN proteins that are important for embryogenesis. We propose that AtCRK5 can govern embryo development in Arabidopsis through the fine tuning of auxin-GA level and the accumulation of certain polar auxin transport proteins.

Assessing the reproducibility of an O-glycopeptide enrichment method with a novel software, Pinnacle.

A novel software, Pinnacle was used to reassess the reproducibility of a 2-step lectin-based O-glycopeptide enrichment method. A publicly available dataset consisting of 12 data files representing 3 technical replicates of enriched glycopeptides from human serum was investigated. Previously, an attempt for reproducibility assessment was made utilizing an MS/MS scan (MS2)-based method. However, the stochastic nature of precursor ion selection strongly biased this approach leading to underestimated rate of reproducibility. To bypass this problem, our present method follows the general path to confidently identify O-glycopeptides (database search with MS/MS data) supplemented with full scan/survey scan (MS1)/extracted ion chromatogram (XIC) mining in all files using two software packages, Pinnacle and Skyline. Confident MS/MS identifications were delivered by Protein Prospector. With this input Skyline indicated a 70% reproducibility for our workflow. However, Pinnacle performed better, indicating the presence of 90% of the confidently assigned glycopeptides in all the three replicates. Pinnacle, just like Skyline, performs ion extraction using the high accuracy, high resolution mass measurement data but it also utilizes all the available MS/MS spectra, even from different activation methods, within the same file to make mass spectrometric data evaluation for glycopeptides more reliable.

Using "spectral families" to assess the reproducibility of glycopeptide enrichment: human serum O-glycosylation revisited.

Growing evidence on the diverse biological roles of extracellular glycosylation as well as the need for quality control of protein pharmaceuticals make glycopeptide analysis both exciting and important again after a long hiatus. High-throughput O-glycosylation studies have to tackle the complexity of glycosylation as well as technical difficulties and, up to now, have yielded only limited results mostly from single enrichment experiments. In this study, we address the technical reproducibility of the characterization of the most prevalent O-glycosylation (mucin-type core 1 structures) in human serum, using a two-step lectin affinity-based workflow. Our results are based on automated glycopeptide identifications from higher-energy C-trap dissociation and electron transfer dissociation MS/MS data. Assignments meeting strict acceptance criteria served as the foundation for generating "spectral families" incorporating low-scoring MS/MS identifications, supported by accurate mass measurements and expected chromatographic retention times. We show that this approach helped to evaluate the reproducibility of the glycopeptide enrichment more reliably and also contributed to the expansion of the glycoform repertoire of already identified glycosylated sequences. The roadblocks hindering more in-depth investigations and quantitative analyses will also be discussed.

Inactivation of plasma membrane-localized CDPK-RELATED KINASE5 decelerates PIN2 exocytosis and root gravitropic response in Arabidopsis.

CRK5 is a member of the Arabidopsis thaliana Ca(2+)/calmodulin-dependent kinase-related kinase family. Here, we show that inactivation of CRK5 inhibits primary root elongation and delays gravitropic bending of shoots and roots. Reduced activity of the auxin-induced DR5-green fluorescent protein reporter suggests that auxin is depleted from crk5 root tips. However, no tip collapse is observed and the transcription of genes for auxin biosynthesis, AUXIN TRANSPORTER/AUXIN TRANSPORTER-LIKE PROTEIN (AUX/LAX) auxin influx, and PIN-FORMED (PIN) efflux carriers is unaffected by the crk5 mutation. Whereas AUX1, PIN1, PIN3, PIN4, and PIN7 display normal localization, PIN2 is depleted from apical membranes of epidermal cells and shows basal to apical relocalization in the cortex of the crk5 root transition zone. This, together with an increase in the number of crk5 lateral root primordia, suggests facilitated auxin efflux through the cortex toward the elongation zone. CRK5 is a plasma membrane-associated kinase that forms U-shaped patterns facing outer lateral walls of epidermis and cortex cells. Brefeldin inhibition of exocytosis stimulates CRK5 internalization into brefeldin bodies. CRK5 phosphorylates the hydrophilic loop of PIN2 in vitro, and PIN2 shows accelerated accumulation in brefeldin bodies in the crk5 mutant. Delayed gravitropic response of the crk5 mutant thus likely reflects defective phosphorylation of PIN2 and deceleration of its brefeldin-sensitive membrane recycling.

O-glycosylation sites identified from mucin core-1 type glycopeptides from human serum.

In this work O-linked glycopeptides bearing mucin core-1 type structures were enriched from human serum. Since about 70 % of the O-glycans in human serum bind to the plant lectin Jacalin, we tested a previously successful protocol that combined Jacalin affinity enrichment on the protein- and peptide-level with ERLIC chromatography as a further enrichment step in between, to eliminate the high background of unmodified peptides. In parallel, we developed a simpler and significantly faster new workflow that used two lectins sequentially: wheat germ agglutinin and then Jacalin. The first lectin provides general glycopeptide enrichment, while the second specifically enriches O-linked glycopeptides with Galß1-3GalNAcα structures. Mass spectrometric analysis of enriched samples showed that the new sample preparation method is more selective and sensitive than the former. Altogether, 52 unique glycosylation sites in 20 proteins were identified in this study.