Tryptic Digestion Of Proteins Research Articles

Label-Free Quantitative Proteomic Analysis of Three Strains of Viscerotropic Leishmania Isolated from Patients with Different Epidemiological Types of Visceral Leishmaniasis in China.

There are three epidemiological types of visceral leishmaniasis in China, which are caused by Leishmania strains belonging to the L. donovani complex. The mechanisms underlying their differences in the population affected, disease latency, and animal host, etc., remain unclear. We investigated the protein abundance differences among Leishmania strains isolated from three types of visceral leishmaniasis endemic areas in China. Promastigotes of the three Leishmania strains were cultured to the log phase and harvested. The protein tryptic digests were analyzed with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), followed by label-free quantitative analysis. The MS experiment was performed on a Q Exactive mass spectrometer. Raw spectra were quantitatively analyzed with the MaxQuant software (ver 1.3.0.5) and matched with the reference database. Differentially expressed proteins were analyzed using the bioinformatics method. The MS analysis wasrepeatedthreetimes for each sample. A total of 5012 proteins were identified across the KS-2, JIASHI-5 and SC6 strains in at least 2 of the three samples replicate. Of them, 1758 were identified to be differentially expressed at least between 2 strains, including 349 with known names. These differentially expressed proteins with known names are involved in biological functions such as energy and lipid metabolic process, nucleotide acid metabolic process, amino acid metabolic process, response to stress, cell membrane/cytoskeleton, cell cycle and proliferation, biological adhesion and proteolysis, localization and transport, regulation of the biological process, and signal transduction. The differentially expressed proteins and their related biological functions may shed light on the pathogenicity of Leishmania and targets for the development of vaccines and medicines.

One-step preparation of cyclen-containing hydrophilic polymeric monolithic materials via epoxy-amine ring-opening reaction and their application in enrichment of N-glycopeptides

Considering the special structure of 1,4,7,10-tetraazacyclododecane (cyclen) which is easy to form complexes with ions, it is beneficial to achieve particular selectivity. Cyclen was selected as a precursor to react with triglycidyl isocyanurate (TGIC), and a novel kind of hydrophilic polymeric monolithic material was facilely prepared via epoxy-amine ring-opening reaction in the presence of a binary porogenic system of acetonitrile (ACN) and polyethylene glycol. The resulting poly (TGIC-co-cyclen) monolithic column was used to separate both nonpolar alkylbenzenes using mobile phase of ACN/H2O (35/65, v/v) and polar phenolic compounds and anilines under the mobile phase of ACN/H2O (60/40, v/v) in reversed-phase capillary liquid chromatography (cLC). It should be pointed that the monolith was further used for separation of a mixture of toluene, DMF, acrylamide and thiourea under the mobile phase of ACN/H2O (95/5, v/v) by hydrophilic interaction chromatography (HILIC). These results indicated that the poly (TGIC-co-cyclen) column exhibited mixed-mode retention mechanism. As a result, the prepared monolithic material was employed for enrichment of glycosylated peptides from the tryptic digest of human immunoglobulin G (IgG) and serum protein tryptic digests. A total of 531 N-glycopeptides and 329 N-glycosylation sites, mapped to 166 glycoproteins, were identified from 2 μL human serum digest. The results indicated the prepared monolith had ability for enriching N-glycopeptides from complex biological samples.

Preparation of zirconium arsenate-modified monolithic column for selective enrichment of phosphopeptides.

Protein phosphorylation is a crucial posttranslational modification for the regulation of many different biological functions. Selective enrichment of phosphopeptides from the complex biological samples is an essential step for the mass spectrometry analysis of protein phosphorylation. In this study, an arsenate functionalized monolithic column was first prepared by a single-step copolymerization of p-methacryloylaminophenylarsonic acid and ethylene dimethacrylate. Then the metal ions Zr4+ were attached onto the prepared monolithic column via metal-chelate complex formation by Zr4+ and arsenate groups. The obtained monolithic column was employed as a new sorbent for the phosphopeptide enrichment via immobilized metal affinity chromatography. Phosphopeptides analysis was realized by polymer monolith microextraction using this monolithic column coupled to both matrix-assisted laser desorption/ionization mass spectrometry and liquid chromatography-electrospray ionization tandem mass spectrometry. The proposed method exhibited a high selectivity for phosphopeptide enrichment in complex matrices, and was applied to the analysis of phosphopeptides in human serum and tryptic digests of rat brain proteins. Four phosphopeptides could be selectively captured from human serum and 2608 endogenous phosphopeptides were identified from the tryptic digests of rat brain proteins, indicating a satisfactory performance of this method for the enrichment of phosphopeptides from complex biological samples.

Capillary electrophoretic profiling of in-bone tryptic digests of proteins as a potential tool for the detection of inflammatory states in oral surgery.

The commonly used histological assessment of pathological states of alveolar bone tissues in oral surgery needs laborious and time-consuming processing by an experienced histologist. Therefore, a simpler and faster methodology is required in this field. Following this demand, this paper reports a straightforward approach using the tryptic cleavage of proteins directly in bone without its demineralization, followed by the capillary electrophoresis-ultraviolet detection profiling of the yielded protein digest. Cleavage-derived peptides were separated by capillary electrophoresis in acidic background electrolytes, pH 2.01-2.54. The best resolution of peptide fragments with the highest peak capacity was achieved in the background electrolyte composed of 55mM H3 PO4 , 14mM tris(hydroxymethyl)aminomethan, pH 2.01. The differences in the obtained capillary electrophoresis-ultraviolet detection profiles with characteristic patterns for particular bone samples were subsequently discriminated by linear discriminant analysis over principal components. This approach was first verified on porcine bone tissues as model samples; jawbone and calf bone tissues could be discriminated with an accuracy of 100%. Subsequently, the method was capable of differentiating unequivocally between human healthy and inflammatory alveolar bone tissues obtained from oral surgery. This procedure seems to be promising as complement or even an alternative to the traditional histological discrimination between healthy and inflammatory bone tissues in oral surgery.

The Seven S Criteria for Evaluating the Performance of a MALDI Mass Spectrometer for MSI

Our goal in this work is to evaluate a new combination linear/reflector MALDI-TOF instrument toward satisfying all "7S criteria" for the ideal MSI mass spectrometer. The linear analyzer satisfies all of the 7 criteria except for Specificity. The new instrument described here adds a reflector to provide up to 50,000 mass resolving power with ppm mass accuracy and with no sacrifice in speed, spatial resolution, and sensitivity demonstrated earlier for the linear MALDI-TOF. This instrument employs new laser optics that produces a 5 kHz laser beam with 2.5-25 μm diameter under computer control. The most important advance is the patented combination of laser and ion optics that provides very high efficiency for production and detection of ions generated by laser desorption using small diameter laser beams. This provides spectra with a wide dynamic range summing a relatively small number of laser shots/pixels. Rat and mouse brain tissues have been used for these initial studies. Examples of negative ion images of lipids and positive ion images from tryptic digestion of proteins are presented. These results demonstrate a very high speed for MSI. This speed is derived from a combination of high laser rate (5 kHz), fast motion of sample relative to the laser beam (20 mm/s), very high ionization efficiency (up to 50%), and the ability to acquire, process, and save spectra at a very high rate (1000/s). As a result, the speed that is possible is imposed by other limits, including the mass range, concentration of samples on the surface, and the spatial resolution required.

Nonspecific cleavages arising from reconstitution of trypsinunder mildly acidic conditions.

Tryptic digestion of proteins followed by liquid chromatography with tandem mass spectrometry analysis is an extensively used approach in proteomics research and biopharmaceutical product characterization, owing to the high level of cleavage fidelity produced with this technique. However, nonspecific trypsin cleavages have been frequently reported and shown to be related to a number of digestion conditions and predigestion sample treatments. In this work, we reveal that, for a number of commercial trypsins, reconstitution and storage conditions can have a significant impact on the occurrence of trypsin nonspecific cleavages. We analyzed the tryptic digestion of a variety of biotherapeutics, using trypsins reconstituted under different conditions. The results indicate that, for many commercial trypsins, commonly recommended reconstitution/storage conditions (mildly acidic, e.g., 50 mM acetic acid, 1 mM HCl) can actually promote nonspecific trypsin activities, which are time dependent and can be as high as 20% in total relative abundance. In contrast, using water for reconstitution and storage can effectively limit nonspecific cleavages to 1%. Interestingly, the performances of different commercial trypsins were found to be quite distinct in their levels of nonspecific cleavages and responses to the two reconstitution conditions. Our findings demonstrate the importance of choosing the appropriate trypsin for tryptic digestion and the necessity of assessing the impact of trypsin reconstitution and storage on nonspecific cleavages. We advocate for manufacturers of commercial trypsins to reevaluate manufacturing processes and reconstitution/storage conditions to provide good cleavage specificity.

Mass Spectrometric Identification of SARS-CoV-2 Proteins from Gargle Solution Samples of COVID-19 Patients.

Mass spectrometry (MS) can deliver valuable diagnostic data that complement genomic information and allow us to increase our current knowledge of the COVID-19 disease caused by the SARS-CoV-2 virus. We developed a simple, MS-based method to specifically detect SARS-CoV-2 proteins from gargle solution samples of COVID-19 patients. The protocol consists of an acetone precipitation and tryptic digestion of proteins contained within the gargle solution, followed by a targeted MS analysis. Our methodology identifies unique peptides originating from SARS-CoV-2 nucleoprotein. Building on these promising initial results, faster MS protocols can now be developed as routine diagnostic tools for COVID-19 patients. Data are available via ProteomeXchange with identifier PXD019423.

Enrichment of histidine containing peptides by on-line immobilised metal affinity solid-phase extraction capillary electrophoresis-mass spectrometry

The analysis and detection of targeted peptides has received due attention in many fields of proteomics research, such as discovery of biomarkers. In this regard, capillary electrophoresis-mass spectrometry (CE-MS) is widely used, however, direct analysis of low-abundance peptides such as histidine-containing peptides (His-peptides) in complex matrixes by CE-MS remains an analytical challenge. In the present study, an immobilised metal affinity solid-phase extraction containing Ni(II) coupled on-line to capillary electrophoresis-mass spectrometry (IMA-SPE-CE-MS) method has been developed to selectively enrich His-peptides from protein tryptic digests and enhance sensitivity. The method was optimised with α-casein and validated with other standard proteins (β-casein and κ-casein). Later, it was applied to an Escherichia Coli (E. Coli) whole cell lysate. IMA-SPE-CE-MS was very selective and allowed an enrichment factor up to 100-fold. The on-line enrichment and separation method coupled to MS detection is straightforward and advantageous over off-line pretreatment methods in terms of simplicity, cost-effectiveness and throughput.

Glutathione-modified ordered mesoporous silicas for enrichment of N-linked glycopeptides by hydrophilic interaction chromatography

Hydrophilic interaction chromatography (HILIC), as one of the important methods for glycopeptides enrichment, has attracted the attention of more and more researchers due to the high efficiency in glycopeptides enrichment. Ordered mesoporous silica materials have been used in many fields, such as catalysis, separation and drug delivery, owing to their adjustable pore size, excellent mechanical properties and ease of preparation. In our case, a series of ordered mesoporous silicas with different pore sizes (3.7–16.7 nm) were prepared via sol-gel reaction employing F127 as the template and TEOS as the silica source. After modified with glutathione by photo-initiated thiol-ene reaction, these materials exhibited hydrophilicity and could be used as adsorbents of HILIC for capturing N-linked glycopeptides from IgG and serum protein tryptic digests. Up to 26 N-glycopeptides were identified from IgG digest, and 565 N-glycopeptides and 330 N-glycosylation sites, mapped to 159 glycoproteins, were identified from 2 μL human serum digest, indicating the great ability for enriching N-glycopeptides from complex biological samples.

Detection and Quantitation of Selected Food Allergens by Liquid Chromatography with Tandem Mass Spectrometry: First Action 2017.17.

In response to a need for accurate and reliable methods for food allergen regulatory compliance, a method for the detection and quantitation of whole egg, whole milk, peanut, and hazelnut in eight food matrices was developed and evaluated in a single-laboratory validation. The matrices include cookies, cookie dough, bread, breakfast cereal, salad dressing, ice cream, and red wine. The method was compared with Standard Method Performance Requirements (SMPR) 2016.002 established by the AOAC Stakeholder Panel on Strategic Food Analytical Methods. The method involves tryptic digestion of allergen proteins in food matrices incurred or spiked with allergen standards [reference materials (RMs), Standard RMs (SRMs), or in-house prepared standard] and uses labeled peptide internal standards. LC-tandem MS analysis of the signature tryptic peptides of the four allergens is performed using multiple reaction monitoring. For 10 allergen/matrix combinations, the method demonstrated adequate sensitivity with a minimum quantitation limit of 3 mg/kg for whole egg and 10 mg/kg for milk, peanut, and hazelnut allergens. Repeatability precision across 3 days of analyses was <17% with analytical range of 10-1000 mg/kg. Recovery from incurred and spiked matrix-matched standards varied from 60 to 118%. The method met the minimum performance requirements of SMPR 2016.002 for whole egg in cookies, bread, cookie dough, and salad dressing; whole milk in cookies and red wine; peanut in breakfast cereal; and hazelnut in cookies. The ERP determined that the data presented met the SMPR and accordingly recommended the method to be granted First Action status. In September 2017, the Official Methods Board approved the method as First Action.

Mass spectrometry-based identification and characterization of human hypothetical proteins highlighting the inconsistency across the protein databases

A myriad of predicted proteins have been described at the genome scale, but their existence has not been confirmed at the protein level. These proteins that are predicted to be expressed from an open-reading frame (ORF) but for which translation has not been demonstrated are known as hypothetical proteins and constitute major fraction of the human proteome. In this study, we aim to identify and characterize hypothetical proteins from human tumor cell lines, viz., HeLa, MCF7, and BT474, thus providing the analytical basis for their expression. We used gel electrophoresis followed by in-gel digestion of the selected protein lanes and subsequent LC–MS/MS analysis of protein tryptic digests. ENSEMBL genome browser was used for genomic alignment. On search against human hypothetical protein data from NCBI database, 110 common proteins were identified across the three selected cells lines. Out of these, 88 proteins were already functionally characterized and remaining 22 were still found to be unreviewed in UniProt, lacking the evidence of expression at the protein level. To explore them further, following HPP guidelines, 15 proteins were selected and aligned against human reference genome. Five hypothetical proteins were confirmed as isoforms of known proteins. We conclude that the proteomic approach used would serve as a suitable tool to validate the existence of predicted or hypothetical proteins at the protein level. The MS proteomics data have been deposited to the ProteomeXchange Consortium via PRIDE with the data set identifiers PXD014258.

Abstract 179: Resistin Regulates ATP-citrate lyase Expression a Key Metabolic Enzyme of Lipogenesis and Histone acetylation in Human Macrophage

BACKGROUND: Human resistin synthesized by macrophages is associated with insulin resistance, type 2 diabetes, atherosclerosis, and chronic inflammation. We used a comparative global protein expression profiling of human macrophage treated with resistin to identify downstream targets and intracellular signaling pathways involving hyper-resistinemia-associated metabolic disorders by HPLC-ESI-MS/MS. ATP-citrate lyase (ACLY), is an essential cytosolic enzyme for generating acetyl-CoA, a key metabolite for glycolysis, de novo lipogenesis, cholesterol synthesis, and histone acetylation. Its dysregulation is associated with diabetes, hypercholesterolemia, and oncogenesis and is a potent therapeutic target . In this study, we explored the regulation of ACLY and its phosphorylation by resistin. METHODS: Mϕ derived from THP1 cells were treated with human resistin for 24-72 hrs. Samples of total protein tryptic digest of untreated control and resistin-treated Mϕ were analyzed by HPLC-ESI-MS/MS. Total protein were also analyzed by western blot for ACLY and its phosphorylation . Gene expression was measured by Quantitative Real-time PCR. RESULTS: ACLY showed a significant (ρ &lt; 0.05) differences in expression between control and resistin treated groups by quantitative mass spectrometry. ACLY formed protein-protein interaction (PPI) with 23 out of 162 significantly differentially regulated proteins. Level of ACLY protein decreased 2 fold after 48 hours of resistin treatment. However, after 72 hours of resistin treatment ACLY proteins increase with a 4-5 fold increase in its serine 455 phosphorylation (Figure 1). In human macrophage, ACLY mRNA also increased ~2 fold after 24 hours of resistin treatment (Figure 2). CONCLUSION: Resistin significantly changed the gene expression of ACLY in humanMϕ. Although there was an initial decrease in ACLY protein with no change in its phosphorylation. However, a longer 72 hours treatment of resistin induce ACLY protein in human macrophages and a very significant increase in its phosphorylation. ACLY plays a pivotal role in many metabolic and epigenetic processes, this late ACLY activation may mediate the effect of resistin on chronic diseases like diabetes, atherosclerosis, and chronic inflammation.

A recommended and verified procedure for in situ tryptic digestion of formalin-fixed paraffin-embedded tissues for analysis by matrix-assisted laser desorption/ionization imaging mass spectrometry.

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is a molecular imaging technology uniquely capable of untargeted measurement of proteins, lipids, and metabolites while retaining spatial information about their location in situ. This powerful combination of capabilities has the potential to bring a wealth of knowledge to the field of molecular histology. Translation of this innovative research tool into clinical laboratories requires the development of reliable sample preparation protocols for the analysis of proteins from formalin-fixed paraffin-embedded (FFPE) tissues, the standard preservation process in clinical pathology. Although ideal for stained tissue analysis by microscopy, the FFPE process cross-links, disrupts, or can remove proteins from the tissue, making analysis of the protein content challenging. To date, reported approaches differ widely in process and efficacy. This tutorial presents a strategy derived from systematic testing and optimization of key parameters, for reproducible in situ tryptic digestion of proteins in FFPE tissue and subsequent MALDI IMS analysis. The approach describes a generalized method for FFPE tissues originating from virtually any source.

Development of amphiphile 4-armed PEO-based Ti4+ complex for highly selective enrichment of phosphopeptides

Protein phosphorylation is a reversible and important post-translational modification. Identification of phosphopeptides without enrichment is difficult for the low-abundance of phosphopeptides in real complex biological samples. Therefore, the effective and selective concentration of phosphopeptides prior to proteomic identification by mass spectrometer is necessary. In this study, we synthesized a novel titanium-based immobilized metal ion affinity chromatography material for highly selective enrichment of phosphopeptides. To improve material hydrophilia to the maximum extent, titanium ions were immobilized on the 4-armed Poly(ethylene oxide)(4μ-PEO-Ti4+), a totally soluble polymer with large molecular weight (20000 g/mol). The 4μ-PEO-Ti4+ was used to enrich phosphopeptides from tryptic digests of standard proteins and real complex biological samples, followed by MALDI-TOF MS analysis. In enrichment of phosphopeptides from 4 pmol β-casein, the 4μ-PEO-Ti4+ performed the best property with starting material of 99–132 μg, loading buffer of 50% ACN/5% TFA (v/v), elution buffer of 10% NH3·H2O (v/v) and elution time of 30 min. The 4μ-PEO-Ti4+ has a superior detection sensitivity as low as 2 fmol for phosphopeptides. The high selectivity of 4μ-PEO-Ti4+ allows a deep enrichment of phosphopeptides of β-casein from a mixture with BSA of 1000-fold abundant. The 4μ-PEO-Ti4+ shows great stability and endurability and can be recycled up to at least 5 times. In addition, 4μ-PEO-Ti4+ could detect 10 and 15 phosphopeptides from non-fat milk and nonenzymatic human saliva, respectively. In total, 4μ-PEO-Ti4+ is a novel excellent material which shows great sensitive and selective enrichment of low-abundance phosphopeptides in real complex biological samples.

Highly selective enrichment of phosphopeptides using poly(dibenzo-18-crown-6) as a solid-phase extraction material.

A poly(dibenzo-18-crown-6) was used as a new solid-phase extraction material for the selective enrichment of phosphopeptides. Isolation of phosphopeptides was achieved based on specific ionic interactions between poly(dibenzo-18-crown-6) and the phosphate group of phosphopeptides. Thus, a method was developed and optimized, including loading, washing and elution steps, for the selective enrichment of phosphopeptides. To assess this potential, tryptic digest of three proteins (α- casein, β-casein and ovalbumin) was applied on poly(dibenzo-18-crown-6). The nonspecific products were removed by centrifugation and washing. The spectrometric analysis was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Highly selective enrichment of both mono- and multiphosphorylated peptides was achieved using poly(dibenzo-18-crown-6) as solid-phase extraction material with minimum interference from nonspecific compounds. Furthermore, evaluation of the efficiency of the poly(dibenzo-18-crown-6) was performed by applying the digest of egg white. Finally, quantum mechanical calculations were performed to calculate the binding energies to predict the affinity between poly(dibenzo-18-crown-6) and various ligands. The newly identified solid-phase extraction material was found to be a highly efficient tool for phosphopeptide recovery from tryptic digest of proteins.

Effects of incubation temperature and acetonitrile amount on microwave-assisted tryptic digestion of proteins

The effects of incubation temperature and acetonitrile (ACN) amount on microwave-assisted tryptic digestion of horse skeletal muscle myoglobin (MYG) and bovine serum albumin (BSA) were investigated. Microwave-assisted tryptic digestion was performed on BSA or MYG solutions containing different amounts (0, 10, and 20%) of ACN for different times (10, 20, 30, 40, and 50 min) at different temperatures (25, 37, and 55 °C). Conventional overnight tryptic digestion was also conducted with gentle mixing at 37 °C for 16 h. Digested samples were analyzed using matrix-assisted laser desorption/ionization mass spectrometry. Similar sequence coverage (SC) values were obtained under most conditions except when the protein sample solutions were digested with 20% ACN at 55 °C, which provided the lowest SC for both MYG and BSA for all investigated digestion times. Considering the missed cleavage (MC) ratios for 50-min microwave-assisted digestion, the highest MC ratio, (i.e., lowest trypsin activity) was observed for the digestion condition of 20% ACN at 55 °C for both proteins, while the lowest MC ratio was observed with 0% ACN at 25 °C for MYG and with 0% ACN at 55 °C for BSA. Conventional overnight tryptic digestion at 37 °C provided more completely cleaved peptides than 50-min microwave-assisted tryptic digestion at the same temperature.

Preparation of epoxy-functionalized hierarchically porous hybrid monoliths via free radical polymerization and application in HILIC enrichment of glycopeptides

Owing to their multiscale pore size regimes and unique properties, the materials with hierarchically porous structures have become an important family of functional materials in recent years. They have been applied from energy conversion and storage, catalysis, separation to drug delivery, etc. The synthesis of them is difficult by the need to employ multiple templates and take complicated steps. Herein, we successfully prepared epoxy-functionalized hierarchically porous hybrid monoliths (HPHMs) with micro/meso/macro-structures in an easy way. Firstly, a bulk monolithic material was formed via free radical polymerization between polyhedral oligomeric vinylsilsesquioxanes (vinylPOSS) and allyl glycidyl ether (AGE) in the presence of polycaprolactone (PCL). Then PCL was degraded with hydrochloric acid solution, and the epoxy-functionalized HPHM was obtained. This approach was very simple and suitable for large-scale preparation. Hybrid monoliths with different specific surface area (from 5.4 to 636.7 m2/g) were prepared by adjusting the mole ratio of vinylPOSS to AGE and the content of PCL. The results of several characterization methods, including nitrogen adsorption/desorption measurements, scanning electron microscopy (SEM) and mercury intrusion porosimetry (MIP), showed that these materials contained not only micropores and mesopores but also macropores. The materials were further modified with penicillamine to be used as hydrophilic interaction chromatography (HILIC) adsorbents for enriching N-glycopeptides in IgG and serum protein tryptic digests. Up to 23 N-glycopeptides were identified from IgG digest, and 385 N-glycopeptides and 283 N-glycosylation sites were identified from human serum digest.

Intact Protein Analysis by LC-MS for Characterizing Biomarkers in Cerebrospinal Fluid.

In the field of proteomics, the emerging "top-down" MS-based proteomics approach can be used to obtain a bird's eye view of all intact proteoforms present in a sample. This alternative to the "bottom-up" approach based on tryptic protein digestion processes has some unique advantages for assessing PTMs and sequence variations. However, it requires some dedicated tools for sample preparation and LC-MS analysis, which makes it more complex to handle than the bottom-up approach. In this study, a simple methodology is presented for characterizing intact proteins in biological fluid. This method yields quantitative information using an MS1 profiling approach and makes it possible to identify the proteins regulated under various clinical conditions.

Core-shell structured magnetic metal-organic framework composites for highly selective enrichment of endogenous N-linked glycopeptides and phosphopeptides

In this work, core-shell structured magnetic metal-organic framework composites denoted as Fe3O4 @MIL-100(Fe) were synthesized by means of a layer-by-layer assembly method selecting Fe as metal center and 1,3,5-benzenetricarboxylic acid as organic ligand. The as-prepared material exhibited outstanding sensitivity (0.1 fmol/μL), good selectivity (1:20 and 1:50 respectively), excellent ability of size-exclusion (1: 500), fine reusability (six cycles) and great stability (two months) in enriching N-linked glycopeptides and phosphopeptides from tryptic digests of standard proteins by combining HILIC and IMAC. Moreover, it was applied into the enrichment of endogenous N-linked glycopeptides and phosphopeptides in human saliva and achieved great results (43 phosphopeptides and 39 N-linked glycopeptides), revealing its promising potential in enrichment of low-abundance endogenous N-linked glycopeptides and phosphopeptides in practical samples.

Temporal Analyses of Barley Malting Stages Using Shotgun Proteomics

Malt derived from barley malting is an essential raw material for beer brewing. In this study, we performed the first dynamic proteome survey during barley malting using a gel-free proteomics approach. This entailed in-solution tryptic digestion of precipitated proteins and analysis of peptides by nanoliquid chromatography coupled with tandem mass spectrometry. A total of 1418 proteins were identified from the five malting stages: Steep, 1, 3, 5 days after germination, and end of Kiln. About 900 proteins identified in this analysis were uncharacterized or predicted proteins. Integrating information from Uniprot90, Uniprot50, Pfam, Interpro databases and gene ontologies from EnsemblPlants, 796 of the predicted and uncharacterized proteins were provided functional annotations. Nearly 63% of the identified proteins were present during all the five time points suggesting a coordinated activation of major metabolic pathways during malting. GO enrichment analysis showed over-representation of proteins associated with translation, carbohydrate metabolism, and stress response. Analysis of variance of the spectral counts of proteins present in all the five malting stages identified 205 proteins with significant differences in their abundance. Proteins associated with carbohydrate metabolism especially enzyme activity regulation provide novel targets for malting barley breeding and for predicting malting quality.