RESUMO
Mucin-type O-glycosylation (or O-GalNAcylation) takes place on most membrane and secretory proteins and is vital in regulating protein functions and many biological processes. O-GalNAcylation generally exhibits highly diverse and dense O-glycans linked to carrier proteins, which challenges the analysis of O-GalNAc glycoproteome using conventional methodologies. Here, we report an O-glycopeptide truncation strategy for the characterization of protein O-GalNAcylation in biological samples. The O-glycopeptide truncation strategy utilizes proteases or O-glycopeptidases for targeted cleavage of the enriched tryptic O-glycopeptides. It simplifies the O-glycopeptide backbones, O-glycans, or both, and has been shown to aid the improvement of the analytical coverage of O-GalNAc glycopeptides and glycoproteins. Tryptic O-glycopeptides covered with O-glycan clusters and terminal sialic acids could be well isolated by the hydrophilic-based enrichment approaches. The enriched O-glycopeptides are then enzymatically truncated into shorter or less multiply O-glycosylated peptides, which are more favorable for mass spectrometry detection and database search in general bottom-up glycoproteomics. We also investigate different proteolysis which could be well integrated into the O-glycopeptide truncation strategy. For large-scale analysis, we exploit different truncation schemes and identify nearly 2000 O-glycopeptides corresponding to 391 glycoproteins from 75 µL human serum, achieving the deepest-scale coverage of O-glycoproteins compared to other plasma/serum O-glycoproteomic studies. Together, the O-glycopeptide truncation strategy has great potential to facilitate the in-depth study of O-GalNAc glycoproteomics in biological samples.
Assuntos
Glicopeptídeos , Proteômica , Humanos , Glicopeptídeos/análise , Proteômica/métodos , Glicoproteínas/química , Glicosilação , Polissacarídeos/análiseRESUMO
MOTIVATION: The interpretation of mass spectrometry (MS) data is a crucial step in proteomics analysis, and the identification of post-translational modifications (PTMs) is vital for the understanding of the regulation mechanism of the living system. Among various PTMs, glycosylation is one of the most diverse ones. Though many search engines have been developed to decipher proteomic data, some of them are difficult to operate and have poor performance on glycoproteomic datasets compared to advanced glycoproteomic software. RESULTS: To simplify the analysis of proteomic datasets, especially O-glycoproteomic datasets, here, we present a user-friendly proteomic database search platform, MS-Decipher, for the identification of peptides from MS data. Two scoring schemes can be chosen for peptide-spectra matching. It was found that MS-Decipher had the same sensitivity and confidence in peptide identification compared to traditional database searching software. In addition, a special search mode, O-Search, is integrated into MS-Decipher to identify O-glycopeptides for O-glycoproteomic analysis. Compared with Mascot, MetaMorpheus and MSFragger, MS-Decipher can obtain about 139.9%, 48.8% and 6.9% more O-glycopeptide-spectrum matches. A useful tool is provided in MS-Decipher for the visualization of O-glycopeptide-spectra matches. MS-Decipher has a user-friendly graphical user interface, making it easier to operate. Several file formats are available in the searching and validation steps. MS-Decipher is implemented with Java, and can be used cross-platform. AVAILABILITY AND IMPLEMENTATION: MS-Decipher is freely available at https://github.com/DICP-1809/MS-Decipher for academic use. For detailed implementation steps, please see the user guide. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Glicopeptídeos , Proteoma , Glicopeptídeos/análise , Glicopeptídeos/química , Proteômica/métodos , Software , Espectrometria de Massas , Peptídeos/químicaRESUMO
Protein tyrosine phosphorylation (pTyr) plays a prominent role in signal transduction and regulation in all eukaryotic cells. In conventional immunoaffinity purification (IP) methods, phosphotyrosine peptides are isolated from the digest of cellular protein extracts with a phosphotyrosine-specific antibody and are identified by tandem mass spectrometry. However, low sensitivity, poor reproducibility, and high cost are universal concerns for IP approaches. In this study, we presented an antibody-free approach to identify phosphotyrosine peptides by using protein tyrosine phosphatase (PTP). It was found that most of the PTPs including PTP1B, TCPTP, and SHP1 can efficiently and selectively dephosphorylate phosphotyrosine peptides. We then designed a workflow by combining two Ti4+-IMAC-based phosphopeptide enrichment steps with PTP-catalyzed dephosphorylation for tyrosine phosphoproteomics analysis. This workflow was first validated by selective detection of phosphotyrosine peptides from semicomplex samples and then applied to analyze the tyrosine phosphoproteome of Jurkat T cells. Around 1000 putative former phosphotyrosine peptides were identified from less than 500 µg of cell lysate. The tyrosine phosphosites on the majority of these peptides could be unambiguously determined for over 70% of them possessing only one tyrosine residue. It was also found that the tyrosine sites identified by this method were highly complementary to those identified by the SH2 superbinder-based method. Therefore, the combination of Ti4+-IMAC enrichment with PTP dephosphorylation provides an alternative and cost-effective approach for tyrosine phosphoproteomics analysis.
Assuntos
Proteômica , Tirosina , Humanos , Peptídeos/química , Fosforilação , Fosfotirosina/química , Proteínas Tirosina Fosfatases , Proteoma/análise , Proteômica/métodos , Reprodutibilidade dos Testes , Tirosina/químicaRESUMO
We present a silicon slot microring resonator for efficient frequency conversion via four-wave mixing (FWM). The slot consists of a narrow silicon waveguide pair with a gap of 80 nm, which is filled with a nonlinear optical polymer. The group velocity dispersion for the microring is controlled by engineering the geometry of the slot structure. Because of the large buildup factor of the slot microring, an FWM conversion efficiency of -27.4 dB is achieved with an optical pump power of less than 1.0 mW. From the measured power dependence of FWM generation, a nonlinear refractive index coefficient of 1.31 × 10-17 m2 W-1 is obtained at a wavelength of 1562 nm. This work presents a hybrid silicon slot and polymer microring as a potential nonlinear device for applications in integrated photonic devices.
RESUMO
Efficient electro-optic (EO) modulation can be generated in the hybrid silicon modulator with EO polymer in the form of an in-plane coplanar waveguide and electrode structure. Strong confinement of the optical field in the hybrid structure is critical to performing efficient electric poling and modulation of the EO polymer. The waveguide consists of silica-based side claddings and an EO core for increasing the integral of the optical field and the overlap interaction between the optical field and the modulated electric field within the EO polymer. We discuss in detail the volume resistivity dependence of the efficiency of electric poling and modulation for various side-cladding materials. In a Mach-Zehnder interferometer modulator, the measured half-wave-voltage length product (VπL) is 1.9 V·cm at an optical communication wavelength of 1,550 nm under the TE optical mode operation. The high-speed signaling of the device is demonstrated by generating on-off-keying transmission at signal rates up to 52 Gbit/s with a Q factor of 6.1 at a drive voltage of 2.0 Vpp.
RESUMO
Cell surface is the primary site for sensing extracellular stimuli. The knowledge of the transient changes on the surfaceome upon a perturbation is very important as the initial changed proteins could be driving molecules for some phenotype. In this study, we report a fast cell surface labeling strategy based on peroxidase-mediated oxidative tyrosine coupling strategy, enabling efficient and selective cell surface labeling within seconds. With a labeling time of 1 min, 2684 proteins, including 1370 (51%) cell surface-annotated proteins (cell surface/plasma membrane/extracellular), 732 transmembrane proteins, and 81 cluster of differentiation antigens, were identified from HeLa cells. By comparison with the negative control experiment using quantitative proteomics, 500 (68%) out of the 731 significantly enriched proteins (p-value < 0.05, ≥2-fold) in positive experimental samples were cell surface-annotated proteins. Finally, this technology was applied to track the dynamic changes of the surfaceome upon insulin stimulation at two time points (5 min and 2 h) in HepG2 cells. Thirty-two proteins, including INSR, CTNNB1, TFRC, IGF2R, and SORT1, were found to be significantly regulated (p-value < 0.01, ≥1.5-fold) after insulin exposure by different mechanisms. We envision that this technique could be a powerful tool to analyze the transient changes of the surfaceome with a good time resolution and to delineate the temporal and spatial regulation of cellular signaling.
Assuntos
Proteoma , Proteômica , Biotinilação , Membrana Celular/metabolismo , Células HeLa , Humanos , Proteoma/metabolismoRESUMO
Converting CO2 and H2 O into carbon-based fuel by IR light is a tough task. Herein, compared with other single-component photocatalysts, the most efficient IR-light-driven CO2 reduction is achieved by an element-doped ultrathin metallic photocatalyst-Ni-doped CoS2 nanosheets (Ni-CoS2 ). The evolution rate of CH4 over Ni-CoS2 is up to 101.8â µmol g-1 h-1 . The metallic and ultrathin nature endow Ni-CoS2 with excellent IR light absorption ability. The PL spectra and Arrhenius plots indicate that Ni atoms could facilitate the separation of photogenerated carriers and the decrease of the activation energy. Moreover, in situ FTIR, DFT calculations, and CH4 -TPD reveal that the doped Ni atoms in CoS2 could effectively depress the formation energy of the *COOH, *CHO and desorption energy of CH4 . This work manifests that element doping in atomic level is a powerful way to control the reaction intermediates, providing possibilities to realize high-efficiency IR-light-driven CO2 reduction.
RESUMO
Endogenous glycopeptides in serum are an invaluable resource for biomarker discovery. Due to the low abundance and the poor fragmentation in tandem mass spectrometry, the identification of endogenously intact glycopeptides still faces many challenges. Herein, an integrated platform is fabricated for the identification of N-linked and O-linked endogenously intact glycopeptides. In this platform, the high-temperature acid denaturation, ultrafiltration, and hydrophilic interaction chromatography steps are combined together for the highly efficient extraction of the endogenously intact glycopeptides from a small amount of serum. Additionally, the twin-spectra scheme and in silico deglycosylation strategy were applied for the identification of N-linked and O-linked endogenous glycopeptides, respectively. In total, 223 intact N-glycopeptides and 51 intact O-glycopeptides are identified from only 40 µL of the human serum sample. This is the first study reporting the identification of endogenously intact N-linked and O-linked glycopeptide and is also the largest data set of endogenously intact glycopeptides reported so far. The distributions of glycans among peptides and proteins and cleavage sites on peptides are further analyzed to seek the regulation of endogenous glycosylation for disease mechanism. The developed strategy provides a novel platform for the disease biomarker discovery.
Assuntos
Glicopeptídeos , Proteômica , Glicopeptídeos/metabolismo , Glicosilação , Humanos , Soro/metabolismo , Espectrometria de Massas em TandemRESUMO
Aberrant sialylation of glycoproteins is closely related to many malignant diseases, and analysis of sialylation has great potential to reveal the status of these diseases. However, in-depth analysis of sialylation is still challenging because of the high microheterogeneity of protein glycosylation, as well as the low abundance of sialylated glycopeptides (SGPs). Herein, an integrated strategy was fabricated for the detailed characterization of glycoprotein sialylation on the levels of glycosites and site-specific glycoforms by employing the SGP enrichment method. This strategy enabled the identification of up to 380 glycosites, as well as 414 intact glycopeptides corresponding to 383 site-specific glycoforms from only initial 6 µL serum samples, indicating the high sensitivity of the method for the detailed analysis of glycoprotein sialylation. This strategy was further employed to the differential analysis of glycoprotein sialylation between hepatocellular carcinoma patients and control samples, leading to the quantification of 344 glycosites and 405 site-specific glycoforms, simultaneously. Among these, 43 glycosites and 55 site-specific glycoforms were found to have significant change on the glycosite and site-specific glycoform levels, respectively. Interestingly, several glycoforms attached onto the same glycosite were found with different change tendencies. This strategy was demonstrated to be a powerful tool to reveal subtle differences of the macro- and microheterogeneity of glycoprotein sialylation.
Assuntos
Carcinoma Hepatocelular/sangue , Glicoproteínas/sangue , Neoplasias Hepáticas/sangue , Proteômica/métodos , Carcinoma Hepatocelular/patologia , Cromatografia Líquida/métodos , Glicopeptídeos/sangue , Glicosilação , Humanos , Neoplasias Hepáticas/patologia , Espectrometria de Massas em Tandem/métodosRESUMO
Tyrosine phosphorylation plays a major role in regulating cell signaling pathways governing diverse biological functions such as proliferation and differentiation. Systemically mapping phosphotyrosine (pTyr) sites is the key to understanding molecular mechanisms underlining pTyr-dependent signaling. Although mass spectrometry-based technologies have been widely used for pTyr site profiling and quantification, their applications are often hindered by the poor efficiency in current multistep enrichment procedures for inherently low abundance pTyr peptides, especially under physiological conditions. Taking advantage of the sequence-independent high affinity of SH2 superbinder toward pTyr residues, we have developed a simplified one-step pTyr peptide enrichment method that uses immobilized SH2 superbinder for unbiased and robust enrichment of endogenous pTyr peptides from biological samples. By eliminating the prerequisite global phosphopeptide enrichment step in our previously developed two-step method, we minimized sample loss and improved peptide capture efficiency. Applying this method to Jurkat cells at resting state, where the tyrosine phosphorylation level is low, both the number of identified pTyr peptides and sites are increased by three folds compared to the two-step method. Specifically, we were able to identify 511 nonredundant pTyr peptides, corresponding to 403 high confidence pTyr sites, from Jurkat cells with high level technical reproducibility (Pearson's correlation coefficient as high as 0.94). Further applying this method to two human breast cancer cell lines, BT474 and HCC1954, before and after EGF stimulation, we demonstrated that this approach could be a powerful tool for illustrating pTyr-dependent signaling network controlling cellular behaviors such as drug resistance.
Assuntos
Fosfopeptídeos , Fosfotirosina , Proteômica/métodos , Domínios de Homologia de src , Linhagem Celular Tumoral , Humanos , Células Jurkat , Fosfopeptídeos/análise , Fosfopeptídeos/química , Fosfopeptídeos/metabolismo , Fosforilação , Fosfotirosina/análise , Fosfotirosina/química , Fosfotirosina/metabolismo , Proteoma/análise , Proteoma/química , Proteoma/metabolismo , Transdução de SinaisRESUMO
For the analysis of homogeneous post-translational modifications such as protein phosphorylation and acetylation, setting a variable modification on the specific residue(s) is applied to identify the modified peptides for database searching. However, this approach is often not applicable to identify intact mucin-type O-glycopeptides due to the high microheterogeneity of the glycosylation. Because there is virtually no carbohydrate-related tag on the peptide fragments after the O-glycopeptides are dissociated in HCD, we find it is unnecessary to set the variable mass tags on the Ser/Thr residues to identify the peptide sequences. In this study, we present a novel approach, termed as O-Search, for the interpretation of O-glycopeptide HCD spectra. Instead of setting the variable mass tags on the Ser/Thr residues, we set variable mass tags on the peptide level. The precursor mass of the MS/MS spectrum was deducted by every possible summed mass of O-glycan combinations on at most three S/T residues. All the spectra with these new precursor masses were searched against the protein sequence database without setting variable glycan modifications. It was found that this method had much decreased search space and had excellent sensitivity in the identification of O-glycopeptides. Compared with the conventional searching approach, O-Search yielded 96%, 86%, and 79% improvement in glycopeptide spectra matching, glycopeptide identification, and peptide sequence identification, respectively. It was demonstrated that O-Search enabled the consideration of more glycan structures and was fitted to analyze microheterogeneity of O-glycosylation.
Assuntos
Glicopeptídeos/análise , Glicopeptídeos/química , Oxigênio/metabolismo , Proteômica/métodos , Sequência de Aminoácidos , Glicopeptídeos/metabolismo , Glicosilação , Fluxo de TrabalhoRESUMO
Many important experiments in proteomics including protein digestion, enzyme substrate screening, enzymatic labeling, etc., involve the enzymatic reactions in a complex system where numerous substrates coexists with an enzyme. However, the enzyme kinetics in such a system remains unexplored and poorly understood. Herein, we derived and validated the kinetics equations for the enzymatic reactions in complex system. We developed an iteration approach to depict the enzymatic reactions in complex system. It was validated by 630 time-course points from 24 enzymatic reaction experiments and was demonstrated to be a powerful tool to simulate the reactions in the complex system. By applying this approach, we found that the ratio of substrate depletion is independent of other coexisted substrates under specific condition. This observation was then validated by experiments. Based on this striking observation, a simplified model was developed to determine the catalytic efficiencies of numerous competing substrates presented in the complex enzyme reaction system. When coupled with high-throughput quantitative proteomics technique, this simplified model enabled the accurate determination of catalytic efficiencies for 2369 peptide substrates of a protease by using only one enzymatic reaction experiment. Thus, this study provided, in the first time, a validated model for the large scale determination of specificity constants which could enable the enzyme substrate screening approach turned from a qualitative method of identifying substrates to a quantitative method of identifying and prioritizing substrates. Data are available via ProteomeXchange with identifier PXD004665.
Assuntos
Enzimas/metabolismo , Peptídeos/análise , Proteômica/métodos , Algoritmos , Ensaios de Triagem em Larga Escala , Cinética , Modelos Teóricos , Especificidade por SubstratoRESUMO
Compared with N-linked glycosylation, the analysis of O-GalNAc glycosylation is extremely challenging due to the high structure diversity of glycans and lack of glycosidases to release O-GalNAc glycans. In this work, a glycoform simplification strategy by combining HILIC enrichment with chemical de-sialylation to characterize O-GalNAc glycosylation of human serum is presented. This method is first validated by using the bovine fetuin as the test sample. It is found that more than 90% of the sialic acid residues can be removed from bovine fetuin by the acid-assisted de-sialylation method, which significantly simplifies the glycan structure and improves identification sensitivity. Indeed, the number of identified peptide backbones increases nearly one fold when this strategy is used. This method is further applied to analyze the human serum sample, where 185 O-GalNAc modified peptide sequences corresponding to 94 proteins with high confidence (FDR (false detection rate) <1%) are identified. This straight forward strategy can significantly reduce the variations of glycan structures, and is applicable to analysis of other biological samples with high complexity.
Assuntos
Acetilgalactosamina/química , Proteínas Sanguíneas/metabolismo , Glicopeptídeos/sangue , Glicoproteínas/sangue , Polissacarídeos/química , Animais , Bovinos , Fetuínas/metabolismo , Glicosilação , Humanos , Polissacarídeos/metabolismo , ProteômicaRESUMO
In this study, we present a method to specifically capture phosphotyrosine (pTyr) peptides from minute amount of sample for the sensitive analysis of protein tyrosine phosphorylation. We immobilized SH2 superbinder on a monolithic capillary column to construct a microreactor to enrich pTyr peptides. It was found that the synthetic pTyr peptide could be specifically enriched by the microreactor from the peptide mixture prepared by spiking of the synthetic pTyr peptide into the tryptic digests of α-casein and ß-casein with molar ratios of 1:1000:1000. The microreactor was further applied to enrich pTyr peptides from pervanadate-treated HeLa cell digests for phosphoproteomics analysis, which resulted in the identification of 796 unique pTyr sites. In contrast, the conventional SH2 superbinder-based method identified 41 pTyr sites for the same sample, only 5.2% of the number achieved by the microreactor. Finally, this microreactor was also applied to analyze the pTyr in Shc1 complex, an immunopurified protein complex, which resulted in the identification of 15 pTyr sites. Together, this technique is best fitted to analyze the pTyr in minute amount of sample and will have broad application in fields where only a limited amount of sample is available.
Assuntos
Proteômica/instrumentação , Tirosina/metabolismo , Desenho de Equipamento , Células HeLa , Humanos , Fosforilação , Fosfotirosina/análise , Proteômica/métodosRESUMO
Human serum is a complex body fluid that contains various N-linked and O-linked glycoproteins. Compared with N-linked glycoproteins, the serum O-linked glycoproteins are not well-studied due to their high heterogeneity and their low abundance. Herein, we presented a novel chemoenzymatic method to analyze core-1 type of O-GalNAcylation in human serum. In this approach, the tryptic digest of serum was first subjected to PNGase F treatment to release the N-glycan and was then treated with strong acid to release sialic acid residues from mucin-type O-glycans. In this way, the internal Gal/GalNAc residues were exposed and were oxidized by the galactose oxidase to carry the aldehyde groups. The oxidized O-GalNAcylated peptides were then captured by hydrazide beads and eluted with methoxylamine for LC-MS/MS analysis. The de-N-deglycosylation decreased the abundance of N-glycopeptides, the desialylation simplified the O-glycans and the enzymatic oxidization conferred the enrichment specificity. We have demonstrated that this method was fitted to analyze O-GalNAcylated peptides with high confidence. This method was applied to analyze human serum, which resulted in the identification of 59 O-GalNAc modified peptide sequences corresponding to 38 glycoproteins from 50 µL of serum. This method is expected to have broad applications in the analysis of O-glycoproteome.
Assuntos
Proteínas Sanguíneas/análise , Glicoproteínas/análise , Proteômica/métodos , Proteínas Sanguíneas/química , Cromatografia Líquida , Galactose Oxidase/química , Glicopeptídeos/análise , Glicopeptídeos/química , Glicoproteínas/química , Glicosilação , Humanos , Hidrólise , Células Jurkat , Oxirredução , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/química , Proteólise , Espectrometria de Massas em Tandem , Tripsina/químicaRESUMO
In this study, we presented an enrichment-free approach for the sensitive analysis of protein phosphorylation in minute amounts of samples, such as purified protein complexes. This method takes advantage of the high sensitivity of parallel reaction monitoring (PRM). Specifically, low confident phosphopeptides identified from the data-dependent acquisition (DDA) data set were used to build a pseudotargeted list for PRM analysis to allow the identification of additional phosphopeptides with high confidence. The development of this targeted approach is very easy as the same sample and the same LC-system were used for the discovery and the targeted analysis phases. No sample fractionation or enrichment was required for the discovery phase which allowed this method to analyze minute amount of sample. We applied this pseudotargeted MS method to quantitatively examine phosphopeptides in affinity purified endogenous Shc1 protein complexes at four temporal stages of EGF signaling and identified 82 phospho-sites. To our knowledge, this is the highest number of phospho-sites identified from the protein complexes. This pseudotargeted MS method is highly sensitive in the identification of low abundance phosphopeptides and could be a powerful tool to study phosphorylation-regulated assembly of protein complex.
Assuntos
Fosfopeptídeos/análise , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/análise , Cromatografia Líquida , Células HeLa , Humanos , Espectrometria de Massas , Fosfopeptídeos/metabolismo , Fosforilação , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/metabolismo , Células Tumorais CultivadasRESUMO
The enzymatic process involving the incubation of a library of substrates with an enzyme is the key step for a few important experiments for bioanalytical chemistry including proteomics analysis, enzymatic labeling, substrate screening, etc. The relationship between the substrates' consumption and their abundances in a complex enzymatic system with a huge number of coexisting substrates of different abundances was not well-known. In this study, we have demonstrated theoretically and experimentally that the priority of substrate consumption depended on their specificity constants but not abundances. We derived the expression between the fractions of the substrates consumed (pi) and their specificity constants. Using the enzymatic system of five synthetic peptide substrates of trypsin, we validated through 24 experiments that the ln(1 - pi) values of competing substrates have linear correlation with their specificity constants, and thus, the priority of substrate depletion has no relation with their abundances. Using a state of the art quantitative proteomics approach, we found that the ln(1 - pi) values of 144 competing substrates between any two of four experiments have a linear relationship and the prioritization of substrates can be achieved by sorting their consumption rates in the experiment. This study will improve our understanding of the enzymatic kinetics in the complex system and will benefit the design of enzymatic analytical approaches.
Assuntos
Modelos Teóricos , Tripsina/metabolismo , Cinética , Peptídeos/metabolismo , Proteômica , Especificidade por SubstratoRESUMO
Sialylation typically occurs at the terminal of glycans, and its aberration often correlates with diseases including neurological diseases and cancer. However, the analysis of glycoprotein sialylation in complex biological samples is still challenging due to their low abundance. Herein, a histidine-bonded silica (HBS) material with a hydrophilic interaction and switchable surface charge was fabricated to enrich sialylated glycopeptides (SGPs) from the digest of proteomics samples. High selectivity toward SGPs was obtained by combining the superior hydrophilicity and switchable-charge characteristics. During the enrichment of sialylated glycopeptides from bovine fetuin digest, seven glycopeptides were detected even at the ratio of 1:5000 with the nonsialylated glycopeptides, demonstrating the high specificity of SGP enrichment by using HBS material. Then, HBS material was further utilized to selectively enrich SGPs from the protein digest of human serum, and 487 glycosites were identified from only 2 µL of human serum; 92.0% of the glycopeptides contained at least one sialic acid, indicating good performance for SGP enrichment by using HBS material. Furthermore, the prepared HBS material also has great potential applications in the analysis of glycoprotein sialylation from other complex biological samples.
Assuntos
Glicoproteínas/sangue , Histidina/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Ácido N-Acetilneuramínico/metabolismo , Dióxido de Silício/metabolismo , Glicoproteínas/metabolismo , Histidina/química , Estrutura Molecular , Ácido N-Acetilneuramínico/sangue , Dióxido de Silício/química , Propriedades de SuperfícieRESUMO
The diversity of O-linked glycan structures has drawn increasing attention due to its vital biological roles. However, intact O-glycopeptides with different glycans are typically not well elucidated using the current methods. In this work, an integrated strategy was developed for comprehensive analysis of O-GalNAc glycosylation by combining hydrophilic interaction chromatography (HILIC) tip enrichment, beam-type collision induced decomposition (beam-CID) detection, and in silico deglycosylation method for spectra interpretation. In this strategy, the intact O-GalNAc glycopeptides were selectively enriched and the original spectra obtained by time-of-flight (TOF)-CID were preprocessed using an in silico deglycosylation method, enabling direct searching without setting multiple glycosylation modifications, which could significantly decrease the search space. This strategy was applied to analyze the O-GalNAc glycoproteome of human serum, leading to identification of 407 intact O-GalNAc glycopeptides from 93 glycoproteins. About 81% of the glycopeptides contained at least one sialic acid, which could reveal the microheterogeneity of O-GalNAc glycosylation. Up until now, this is the largest data set of intact O-GalNAc glycoforms from complex biological samples at the proteome level. Furthermore, this method is readily applicable to study O-glycoform heterogeneity in other complex biological systems.
Assuntos
Proteínas Sanguíneas/metabolismo , Glicopeptídeos/sangue , Proteômica/métodos , Animais , Bovinos , Cromatografia Líquida de Alta Pressão , Fetuínas/metabolismo , Glicosilação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Espectrometria de Massas em TandemRESUMO
Protein methylation is receiving more and more attention for its important regulating role in diverse biological processes including epigenetic regulation of gene transcription, RNA processing, DNA damage repair, and signal transduction. Global analysis of protein methylation at the proteome level requires the enrichment of methylated peptides with various forms; unfortunately, the immunoaffinity purification method can only enrich a subset of them due to lacking of pan specific antibody. Because methylation does not significantly alter the physicochemical properties of arginine or lysine residues, chemical approach for global methylome analysis is still at infancy. In this study, by exploiting the fact that the methylation on Arg and Lys prohibiting the cleavage by proteases for these sites, we developed an antibody-free method to enrich methylated peptides, which enabled the identification of 887 methylation forms on 768 sites from HepG2 cells. This technique allows the simultaneous analysis of both Lys and Arg methylation while it has better performance for the identification of Arg methylation. It should find broad applications in studying methylation regulated biological processes.