BSA Digest Research Articles

An explore method for quick screening biomarkers based on effective enrichment capacity and data mining

Protein glycosylation acts as a crucial role in regulating protein function and maintaining cellular homeostasis. Efficient peptide enrichment can be utilized to effectively solve the inherent challenges of protein glycosylation analysis to search unknown cancer biomarkers. In this research, a low dimensional porous hydrophilic nanosheets with a multi-level porous structure (Co-MOF-SiO2@HA) was synthetized via an easy one-pot method for the efficient enrichment of the N-glycopeptides in the digests of complex biosamples. The synthetized nanosheets Co-MOF-SiO2@HA demonstrated excellent enriching performances including a high enrichment capacity (300 mg g-1 calculated), a spectacular selectivity (IgG digests and BSA digests at the molar ratio of 1/1200), and an excellent spatial confinement ability (IgG digests, IgG and BSA at the molar ratio of 1/1000/1000). As an explore result, after the enrichment of human colorectal cancer tissue and human healthy tissue by the nanosheets, several proteins related to cancers and one protein directly related to well-known human colorectal cancer were identified by detecting the corresponding glycopeptides. It presented the potential value of the feasibility of this analysis mode by nanosheets Co-MOF-SiO2@HA in proteomic analysis.

Multiply-mesoporous hydrophilic titanium dioxide nanohybrid for the highly-performed enrichment of N-glycopeptides from human serum

N-glycopeptide is considered as one of significant biomarkers which provide guidance for the diagnosis and drug design of diseases. However, the direct analysis of N-glycopeptides is nearly impracticable mainly owing to their extremely low abundance and grave signal suppression from other interfering substances in the bio-samples. In this research, a multiply-mesoporous hydrophilic TiO2 nanohybrid (mM-TiO2@Cys) was synthesized by immobilizing Cys on a TiO2 substrate with hierarchical mesopores to achieve the highly-performed enrichment of N-glycopeptides. With the advantages of superior hydrophilicity and multiply-mesoporous structure, the obtained material exhibited an excellent selectivity (IgG digests and BSA digests at the molar ratio of 1/500), a high sensitivity (1 fmol μL−1 for IgG digests) and a good size-exclusion ability (IgG digests, IgG and BSA at the molar ratio of 1/500/500) in the enrichment of N-glycopeptides from IgG digests. As a result, 281 N-glycopeptides corresponded with 109 glycoproteins were identified from 2 μL serum digests of the patients with nasopharyngeal carcinoma, and 181 N-glycopeptides corresponded with 78 glycoproteins were identified from 2 μL serum digests of the healthy volunteers, revealing the potential application value of mM-TiO2@Cys in glycoproteomics.

Facile fabrication of hydrophilic covalent organic framework composites for highly selective enrichment of N-glycopeptides

The development of facilely synthetic materials acts an essential role in glycoproteome analysis, especially for the highly efficient enrichment of N-linked glycopeptides. In this work, a facile and timesaving route was introduced in which COFTP-TAPT served as a carrier and poly (ethylenimine) (PEI) and carrageenan (Carr) were successively coated on the surface via electrostatic interaction. The resultant COFTP-TAPT@PEI@Carr showed remarkable performance in glycopeptide enrichment with high sensitivity (2 fmol μL−1), high selectivity (1:800, molar ratio of human serum IgG to BSA digests), large loading capacity (300 mg g−1), satisfactory recovery (102.4 ± 6.0%) and reusability (at least eight times). Due to the brilliant hydrophilicity and electrostatic interactions between COFTP-TAPT@PEI@Carr and positively charged glycopeptides, the prepared materials could be applied in the identification and analysis in the human plasma of healthy subjects and patients with nasopharyngeal carcinoma. As a result, 113 N-glycopeptides with 141 glycosylation sites corresponding to 59 proteins and 144 N-glycopeptides with 177 glycosylation sites corresponding to 67 proteins were enriched from 2 μL plasma trypsin digests of the control groups and patients with nasopharyngeal carcinoma, respectively. 22 glycopeptides were identified only from the normal controls and 53 glycopeptides were detected only from the other set. The results demonstrated that this hydrophilic material was promising on a large scale and further N-glycoproteome research.

Enhancing Biomolecule Analysis and 2DMS Experiments by Implementation of (Activated Ion) 193 nm UVPD on a FT-ICR Mass Spectrometer.

Ultraviolet photodissociation is a fast, photon-mediated fragmentation method that yields high sequence coverage and informative cleavages of biomolecules. In this work, 193 nm UVPD was coupled with a 12 Tesla FT-ICR mass spectrometer and 10.6 μm infrared multi-photon dissociation to provide gentle slow-heating of UV-irradiated ions. No internal instrument hardware modifications were required. Adjusting the timing of laser pulses to the ion motion within the ICR cell provided consistent fragmentation yield shot-to-shot and may also be used to monitor ion positions within the ICR cell. Single-pulse UVPD of the native-like 5+ charge state of ubiquitin resulted in 86.6% cleavage coverage. Additionally, IR activation post UVPD doubled the overall fragmentation yield and boosted the intensity of UVPD-specific x-type fragments up to 4-fold. This increased yield effect was also observed for the 6+ charge state of ubiquitin, albeit less pronounced. This indicates that gentle slow-heating serves to sever tethered fragments originating from non-covalently linked compact structures and makes activation post UVPD an attractive option to boost fragmentation efficiency for top-down studies. Lastly, UVPD was implemented and optimized as a fragmentation method for 2DMS, a data-independent acquisition method. UVPD-2DMS was demonstrated to be a viable method using BSA digest peptides as a model system.

Terpolymeric platform with enhanced hydrophilicity via cysteic acid for serum intact glycopeptide analysis.

A new polymeric (methyl methacrylate/ethylene glycol dimethacrylate/1,2-epoxy-5-hexene) base/matrix has been fabricated and decorated with zwitterionic hydrophilic cysteic acid (Cya) for the enrichment of intact N-glycopeptides from standards and biological samples. Terpolymer-Cya provides good enrichment efficiency, improved hydrophilicity, and selectivity by virtue of better surface area (2.09 × 102 m2/g) provided by terpolymer and the zwitterionic property offered by cysteic acid. Cysteic acid-functionalized polymeric hydrophilic interaction liquid chromatography (HILIC) sorbent enriches 35 and 24N-linked glycopeptides via SPE (solid phase extraction) mode from tryptic digests of model glycoproteins, i.e., immunoglobulin G (IgG) and horseradish peroxidase (HRP), respectively. Zwitterionic chemistry of cysteine helps in achieving higher selectivity with BSA digest (1:200), and lower detection limit down to 100 attomoles with a complete glycosylation profile of each standard digest. The recovery of 81% and good reproducibility define the application of terpolymer-Cya for complex samples like a serum. Analysis of human serum provides a profile of 807 intact N-linked glycopeptides via nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS). To the best of our knowledge, this is the highest number of glycopeptides enriched by any HILIC sorbent. Selected glycoproteins are evaluated in link to various cancers including the breast, lung, uterine, and melanoma using single-nucleotide variances (BioMuta). This study represents the complete idea of using an in-house developed strategy as a successful tool to help analyze, relate, and answer glycoprotein-based clinical issues regarding cancers.

Fabrication of hydrophilic titanium (IV)-immobilized polydispersed microspheres via inverse suspension polymerization for enrichment of phosphopeptides in milk

It is prerequisite to efficient extraction of phosphopeptides in bottom-up strategy for protein phosphorylation research. A kind of Ti4+-immobilized polydispersed phosphate-rich microsphere was fabricated via inverse suspension polymerization by employing vinylphosphonic acid (VPA) and N,N-methylenebisacrylamide (MBA) as functional monomer and crosslinker, respectively. The resulting microsphere demonstrated excellent sensitivity (as low as 10 fmol), selectivity (the mass ratio of β-casein to BSA digests is 1/500), and adsorption capacity (up to 200 mg g−1). What's more, 113 unique phosphopeptides assigned to 25 unique phosphoproteins were indiscriminately identified from 5 μL of pasteurized milk digest, exhibiting great performance in capturing phosphopeptides. In this approach, only two steps were required to synthesize Ti4+-IMAC (Ti4+-Immobilized metal affinity chromatography). Compared with other methods required multistep modifying process, this strategy is simple and time-saving, offering a prospect of pilot production and commercialization. It is expected that the application of IMAC in milk and other food samples will still make it possible to unravel the huge complexity of the Foodome in the near future.

Proteolytic Sensitivity, In Vitro Glycation Efficiency of Diabetic and Non-diabetic Human Serum Albumin

Background: Glycation of human serum albumin (HSA) leads to disturbances in its stability, activity, and other properties which, in turn, affect the functional properties of HSA. Modification of albumin by glycation shows considerable potential as a significant biochemical biomarker for diagnosing diabetes. The characteristics of the glycation process in proteins have not been fully examined yet and, therefore, there is insufficient knowledge about them in the field. Objectives: This study aimed to clarify the differences between diabetic and non-diabetic HSA as well as their structure-function relationship. Methods: The physiological and laboratory characteristics of glycated albumin as well as HSA were explored. A total of 30 subjects were enrolled in this study in which 15 normal healthy individuals were assigned into the control group, and 15 type-2 diabetic patients were included in the diabetic group. Patients with type-1 diabetes, pregnant women, and individuals with other diseases were excluded from the study. Protein estimation, polyacrylamide gel electrophoresis, ammonium sulphate fractionation, dialysis, glycation of HSA followed by gel electrophoresis of glycated samples, digestion of BSA, as well as HSA by α-chymotrypsin and their documentation and stoichiometry were all performed. Results: Various characteristic differences were observed between diabetic and non-diabetic HSA including proteolytic susceptibility and in vitro glycation efficiency. Hypoalbuminemia was, particularly, observed in diabetic patients, which was suggestive of a relationship between hyperglycemia and hypoalbuminemia. Conclusion: Peculiar contrariety between diabetic and non-diabetic HSA, specific differences in their glycation efficiencies, as well as proteolytic susceptibility and their innuendos were precisely traced out. It was concluded that albumin may have been regarded as a significant clinical biomarker for diagnosing diabetes.

Construction of boric acid-functionalized metal-organic frameworks for glycopeptide recognition in the serum of cervical cancer patients.

Cervical cancer is one of the most common malignant tumors in women, and it is essential to explore potential biomarkers such as glycopeptides closely related to cancer in physiological samples of cervical cancer patients. Sample pretreatment is required before direct detection using mass spectrometry because there are certain limitations. Meanwhile, it is still highly desired to promote the functionalization and application of metal-organic framework (MOF)-derived materials. Using a post-synthesis modification method, a novel type of boric acid-functionalized MOF probe (designated as UiO-66@PEI@Au@B(OH)2 ) is prepared for recognition of glycopeptides. The results are obtained using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nano-liquid chromarography-tandem mass spectrometry. The UiO-66@PEI@Au@B(OH)2 probe exhibits a low detection limit (0.6fmol μL-1 ), an excellent recovery rate, comparatively good reusability and selectivity (HRP digests:BSA digests =1:500). When UiO-66@PEI@Au@B(OH)2 is used to selectively capture glycopeptides from the serum of a healthy person and a cervical cancer patient, 101 glycopeptides corresponding to 54 glycoproteins and 108 glycopeptides corresponding to 57 glycoproteins are detected, respectively. The successful preparation of UiO-66@PEI@Au@B(OH)2 provides a path for the investigation of the functionalization of MOF-derived materials. The excellent performance of UiO-66@PEI@Au@B(OH)2 not only demonstrates the huge potential of functionalized MOFs in the glycoproteome, but also opens up new phases of the application of MOF-based materials.

Hydrophilic arginine-functionalized mesoporous polydopamine-graphene oxide composites for glycopeptides analysis

Considering the importance of glycopeptides in the clinical diagnosis of cancer and some serious diseases, the identification of glycopeptides from complex biological samples has attracted considerable attention. Effective pre-enrichment before mass spectrometry analysis plays an important role. In this work, a kind of hydrophilic two-dimensional composites (denoted as GO@MPDA@Arg) based on mesoporous polydopamine-graphene oxide were used to selectively enrich glycopeptides in biological samples. The mesoporous polydopamine (MPDA) layer self-assembled with template Pluronic F127 provided more binding sites to load arginine, and bound arginine enhanced the hydrophilicity of the material. As a result, GO@MPDA@Arg composites exhibited excellent enrichment performance for glycopeptides, containing good selectivity (IgG digests : BSA digests = 1:50, molar ratio), low detection limit for IgG digests (10 fmol μL−1), high loading capacity for IgG digests (200 μg mg−1), and good size exclusion (IgG digests : IgG : BSA = 1:100:100, mass ratio). In addition, mouse brain tissue was selected as the actual biological sample to further study the enrichment effect of GO@MPDA@Arg composites. In three parallel experiments, a total of 401 glycopeptides belonging to 233 glycoproteins were enriched from 200 μg digestion of mouse brain extract. The enrichment results demonstrate that GO@MPDA@Arg composites have application potential for glycopeptides enrichment in protein post-translational modification research.

Post-synthesis of boric acid-functionalized magnetic covalent organic framework as an affinity probe for the enrichment of N-glycopeptides.

A novel type of boric acid-functionalized magnetic covalent organic framework (mCOF) with polyethyleneimine (PEI) as a linker (denoted as mCOF@PEI@B(OH)2) has beenprepared through a post-synthesis strategy, which points out an achievable path for the construction of boronic acid-functionalized COFs. Based on the boric acid chemistry, the obtained core-shell structured mCOF@PEI@B(OH)2 can selectively isolate glycopeptides through the modified boronic acid groups. The mCOF@PEI@B(OH)2 exhibits excellent performance with good reusability (ten cycles), low detection limit (0.5 fmol·μL-1), size-exclusion effect, and relatively high loading capacity (80μg·mg-1), recovery yield (94.9 ± 2.8%), and selectivity (HRP digests:BSA digests = 1:500). Detection is done bymatrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In addition, 37 endogenous glycopeptides are captured from human saliva with mCOF@PEI@B(OH)2, providing effective proofs for its capability to capture low-abundance glycopeptides from actual biological samples.

Flexible and hierarchical metal-organic framework composite as solid-phase media for facile affinity-tip fabrication to selectively enrich glycopeptides and phosphopeptides

Micro-tip-based solid-phase microextraction is considered as one of the green and powerful analytical sample preparation techniques, but its efficiency is severely hampered by some basic issues such as tedious fabrication, instability of sorbent bed, and blocking of the tip, especially for biological samples due to low permeability. These issues are tackled by introducing a flexible and hierarchical substrate in the microtip, having good mechanical strength and specific functionality to capture the desired biomolecules. Considering the well-ordered and flexible structure of melamine foam, it was used as a substrate and for hydrophilic interaction chromatography (HILIC). Metal-organic framework, due to its excellent characteristics, was grafted on its surface anchored by self-assembling polydopamine. The resulting material was characterized and packed in the tip by just pressing the material in the conical structure of the tip. This affinity tip established good and tunable permeability and was used to selectively enrich glycopeptides as well as phosphopeptides. The affinity tip demonstrated excellent performance to enrich glycopeptides and phosphopeptides with a low limit of detection up to 0.5 fmol μL−1 from tryptic digests of horseradish peroxidase and β-Casein, respectively, and was stable up to 5 rounds of enrichment. Moreover, this affinity-tip also exhibited high selectivity up to up to 1:1000 (HRP digest to BSA digest) for glycopeptides and 1:200 (β-Casein digest to BSA digest) for phosphopeptides and demonstrated several other fascinating characteristics such as; excellent size exclusion effect for the omission of large-sized proteins, modest backpressure, reproducibility, reusability, smooth enrichment, and successfully applied to a human saliva sample.

Melamine foam assisted in-tip packed amine-functionalized titanium metal–organic framework for the selective enrichment of endogenous glycopeptides

Endogenous glycopeptides are significantly important in diverse pathological and physiological systems, but their direct analysis is severely hampered by their low abundance and presence of interfering species in biological fluids. In this study, we synthesized the amine-functionalized titanium metal-organic framework (NH2-MIL-125(Ti)) by a simple hydrothermal method, characterized and used for glycopeptides enrichment. The designed separation media is highly hydrophilic and stable which is suitable for hydrophilic interaction chromatography (HILIC). To make the process smooth, simple, reliable, and robust, NH2-MIL-125(Ti) crystals were packed in pipette-tip using hydrophilic melamine foam, as supporting frit. Free amine groups, present in the structure imparted hydrophilicity and a unique pattern of porosity, contributing to the size exclusion effect that excluded the large-sized proteins up to 1:700 peptide to protein ratio. The prepared MOF particles possessed regular porosity, high surface area, good hydrophilicity, and offered an in-tip flow-based set-up enhanced the enrichment performance for N-linked glycopeptides. The affinity material showed a detection limit of 1 fmol.µL−1 and selectivity up to 1:1000 (HRP digest to BSA digest). Moreover, repeatability and reusability were evaluated up to five rounds of enrichment using the same affinity tip, and scanning electron microscopic images revealed no structural changes in the MOF crystals. Finally, the MOF packed in pipette tip was applied to selectively capture the N-linked endogenous glycopeptides from a healthy saliva sample and 64 unique endogenous glycopeptides were identified. These results demonstrated the excellent potential of NH2-MIL-125(Ti) based affinity tip for glycopeptides which can be used to enrich trace glycopeptide biomarkers from the biological fluids.

Recognition of urinary N-linked glycopeptides in kidney cancer patients by hydrophilic carbohydrate functionalized magnetic metal organic framework combined with LC-MS/MS.

A hydrophilic carbohydrate functionalized magnetic metal organic framework (Mag Zr-MOF@G6P) was synthesized via a facile one-step modification strategy for selective glycopeptide capture in virtue of hydrophilic interaction chromatography technique. The inherently hydrophilic Zr-MOF layer not only provides selective size-sieving pore structures but also offers large specific surface area to afford abundant affinity sites. Hydroxyl-rich glucose-6-phosphate was immobilized onto the Zr-MOF via a straightforward coordination manner to regulate its surface property, for the purpose of enhancing its hydrophilicity. Benefitting from the merits of Zr-MOF and glucose-6-phosphate, the as-designed composite exhibits good selectivity (the mass ratio of HRP digests to BSA digests was up to1:200) and low limit of detection (0.1fmolμL-1) towards the recognition of glycopeptides from standard samples. More excitingly, glycopeptides in urine of healthy people and patients with kidney cancer were successfully enriched and identified by the combined liquid chromatography-mass spectrometry/mass spectrometry technology (LC-MS/MS). Further gene ontology analysis of molecular function and biological process revealed that 13 original glycoproteins of the identified glycopeptides from urine of patients significantly participate in diverse cancer-associated events, including collagen binding, immunoglobulin receptor binding, antigen binding, and complement activation process. Graphical abstract.

Preparation and application of peptide molecularly imprinted material based on mesoporous metal-organic framework

In this study, a new molecularly imprinted material, MIP@UiO-66-NH2, was synthesized with glutathione (GSH) as template and mesoporous metal organic framework (UiO-66-NH2) as matrix. The molecularly imprinted polymer was modified on the surface and into the pores of the UiO-66-NH2 by surface molecular imprinting method with thin polymer layer. Based on high specific surface area (1091.93 m2 g−1) and appropriate pore size (35 nm) of the ordered mesoporous UiO-66-NH2, the adsorption capacity for GSH reached 94.43 mg g−1, and the adsorption equilibrium could be achieved within 30 min. The adsorption isotherm data of MIP@UiO-66-NH2 could be described well by Freundlich model and the kinetic data complied well with pseudo-second-order model. In addition, the MIP@UiO-66-NH2 showed low adsorption capacity to GSH structural analogs (QL-cys = 6.51 mg g−1), suggesting great selectivity for GSH recognition. Finally, the MIP@UiO-66-NH2 was successfully applied for selective separation of GSH from BSA, skim milk and egg white tryptic digest.

Highly porous terpolymer-ZIF8@BA MOF composite for identification of mono- and multi-glycosylated peptides/proteins using MS-based bottom-up approach.

A hydrophilic terpolymer MOF composite is designed with high surface area and porosity to enrich mono- and multi-glycosylated peptides facilitating a bottom-up approach. Terpolymer@ZIF-8 is synthesized using free radical polymerization followed by layer by layer ZIF-8 fabrication. Subsequent surface modification was made by aminophenylboronic acid (AMBA). The enrichment ability of terpolymer@ZIF-8@BA is evaluated by using tryptic digest of IgG and HRP to exemplify mono- and multi-glycosylated protein samples. Improved selectivity of 1:200 for spiked HRP in BSA digest and sensitivity down to 1fmolμL-1 is achieved. Batch to batch reproducibility is better 1% RSD which favors the adoption of the developed method for routine N-linked glycopeptide/protein determination. Cost-effective nature of given approach is given by regeneration of the material up to four cycles. Total 318 N-linked glycopeptides have been identified from 1μL human serum digest after subjecting the enriched and PNGase-treated deglycosylated peptides to LC-MS. Thus, terpolymer@ZIF-8@BA holds the potential both for mono- and multi-glycosylated peptides from complex biological sample. Graphical abstract.

Magnetic titanium dioxide nanomaterial modified with hydrophilic dicarboxylic ligand for effective enrichment and separation of phosphopeptides and glycopeptides

A dual functionalized magnetic nanomaterial was fabricated by combining the magnetic core, titania shell, and hydrophilic molecules (denoted as Fe3O4@TiO2-IDA). Based on the mechanism of metal oxide affinity chromatography and hydrophilic interaction liquid chromatography, the sorbent shows excellent one-step separation capacity for both glycopeptides and phosphopeptides. For phosphopeptide enrichment, Fe3O4@TiO2-IDA exhibits high sensitivity and selectivity. The concentration of β-casein in the digests can be as low as 0.0125ngμL-1; the mass ratio of β-casein and BSA digest can reach 1:800. For glycopeptide enrichment, Fe3O4@TiO2-IDA also exhibits good performance. The concentration of horseradish peroxidase (HRP) in the digested sample can be as low as 0.04ngμL-1; the mass ratio of HRP and BSA digest can reach 1:100. Following the one-step enrichment, elution, and nano LC-MS/MS analysis, 550 unique phosphopeptides and 330 glycopeptides were identified from 100μg mouse brain sample through asingle run of LC-MS/MS. Graphical abstract Multipurpose Fe3O4@TiO2-IDA nanomaterials are employed in simultaneous enrichment and separation of glycopeptides and phosphopeptides.

Development of a novel sheathless CE-ESI-MS interface via a CO2 laser ablated opening

A new and easy to construct sheathless capillary electrophoresis electro spray ionization mass spectrometry (CE-ESI-MS) interface was developed that offers several advantages compared to traditional liquid junction interfaces. The fabrication of the device only requires a CO2 laser engraver that most groups working with microfluids have access to. It only takes a few seconds to create a CO2 laser ablated opening in the bare-fused silica capillaries and the opening can be placed as close as a few mm from the spray tip. The capillary is punctured through a silicone tube such that the opening is directly placed inside this tube, which also serves as a liquid reservoir for the make-up liquid. Electrical contact required for both CE separation and ESI is established via the liquid in this reservoir which is in contact with the electrode of an external high voltage power supply. The developed CE-ESI-MS interface is capable of analysing both small molecules and biomolecules such as peptides in physisorbed PEG polymer brush coated capillaries. Proof-of-principle of the interface was demonstrated by analysing a tryptic digest of BSA. Further, a range of drugs of abuse were also investigated. The examined small molecules (pethidine, nortriptyline, methadone, haloperidol and loperamide) have a quantification limit (LOQ) of 150 ng/mL and a detection limit (LOD) of 40 ng/mL (except for loperamide: LOD = 80 ng/mL). Finally, we used our novel CE-MS interface for the analysis of the Aβ40 peptide. This is a member of the beta-Amyloid peptide family, involved in the development of Alzheimer's disease. A LOQ of 9 μg/mL was obtained for Aβ40, corresponding to 23 fmoles in a sample volume of 11 nL.

Reduced surface adsorption in 3D printed acrylonitrile butadiene styrene micro free-flow electrophoresis devices.

We have 3D printed and fabricated micro free-flow electrophoresis (µFFE) devices in acrylonitrile butadiene styrene (ABS) that exhibit minimal surface adsorption without requiring additional surface coatings or specialized buffer additives. 2D, nano LC-micro free flow electrophoresis (2D nLC × µFFE) separations were used to assess both spatial and temporal broadening as peaks eluted through the separation channel. Minimal broadening due to wall adsorption was observed in either the spatial or temporal dimensions during separations of rhodamine 110, rhodamine 123, and fluorescein. Surface adsorption was observed in separations of Chromeo P503 labeled myoglobin and cytochrome c but was significantly reduced compared to previously reported glass devices. Peak widths of <30 s were observed for both proteins. For comparison, Chromeo P503 labeled myoglobin and cytochrome c adsorb strongly to the surface of glass µFFE devices resulting in peak widths >20min. A 2D nLC × µFFE separation of a Chromeo P503 labeled tryptic digest of BSA was performed to demonstrate the high peak capacity possible due to the low surface adsorption in the 3D printed ABS devices, even in the absence of surface coatings or buffer additives.

Preparation of zwitterionic cysteine-modified silica microsphere capillary packed columns for the on-column enrichment and analysis of glycopeptides in human saliva

In this work, for the first time, L-cysteine (L-Cys), a zwitterionic hydrophilic compound containing abundant amino and carboxyl was modified on silica microspheres for the fabrication of SiO2@L-Cys capillary packed column. Since the SiO2@L-Cys capillary packed column possessed great hydrophilicity brought by L-cysteine, they could be utilized to enrich glycopeptides with a low detection limit of 5 fmol μL−1 HRP digests as well as a good selectivity of the mixture of HRP and BSA digests up to a molar ratio of 1:50. The capillary packed column were further applied into the glycosylation analysis of human saliva and 69 glycopeptides were identified from 2 μL human saliva digests.

Site-Specific Quantification of Persulfidome by Combining an Isotope-Coded Affinity Tag with Strong Cation-Exchange-Based Fractionation.

Protein persulfidation is one of the most important oxidative translational modifications and plays vital roles in various important biological processes. However, the proteome-wide identification of persulfidation sites is a great challenge because of the difficulties in accurately differentiating persulfide groups with disulfide and thiol groups in proteins as well as the extremely low abundance of persulfidated peptides. By current approaches, the persulfidated peptides were often identified by the cleavage of their persulfide groups by reductants prior to MS analysis; therefore, it would bring about a false positive identification and was unable to identify persulfidation sites accurately for a single peptide with multiple cysteine residues. In this study, a novel strategy for the site-specific quantification of persulfidome (SSQPer) was developed. By this strategy, the persulfidated proteins were first labeled with cleavable isotope-coded affinity tag (c-ICAT) reagents. After digestion, the labeled persulfidated peptides were selectively enriched with streptavidin beads and fractionated by strong cation exchange chromatography, followed by LC-MS/MS identification. To evaluate the performance of SSQPer, the persulfidated BSA digests with 20 persulfidation sites identified were used to spike HeLa cell digests with mass ratios of 1:100 and 1:1000, and 16 and 13 persulfidated sites were respectively identified. We applied SSQPer to the site-specific quantification of persulfidome in the epithelial-mesenchymal transition (EMT) process, and 226 endogenous persulfidation sites were identified, of which 74.3% were newly discovered. All of these results demonstrated that the SSQPer strategy would provide a promising tool to profile the site-specific persulfidome and pave the way for future investigation to expand our knowledge of persulfidation.