Human serum N-glycome profiling via the newly developed asparagine immobilized cellulose/polymer nanohybrid.

Human serum N-linked glycans expression levels change during the disease progression. The low abundance, structural diversity, and coexisting matrices hinder their detection in mass spectrometry analysis. Considering the hydrophilic nature of N-glycans, cellulose/polymer (1,2-Epoxy-5-hexene) nanohybrid is fabricated with oxirane groups functionalized of asparagine to develop solid phase extraction based hydrophilic interaction liquid chromatography sorbent (cellulose/1,2-Epoxy-5-hexene/asparagine). The morphology, elemental analysis, and surface properties are studied through scanning electron microscopy, energy dispersive X-ray spectroscopy, and Fourier-transform infrared spectroscopy. The large surface area of cellulose/polymer nanohybrid (2.09 × 102  m2 /g) facilitates the high density of asparagine immobilization resulting in better hydrophilic interaction liquid chromatography enrichment under optimized conditions. The enrichment capability of nanohybrid/asparagine is assessed by the N-Linked glycans released from ovalbumin and immunoglobulin G where 23 and 13 N-glycans are detected respectively. The nanohybrid/asparagine shows selectivity of 1:1200 with spiked bovine serum albumin and sensitivity down to 100 attomole. Human serum profiling for N-glycans identifies 52 glycan structures. This new enrichment strategy enriches serum N-linked glycans in the presence of salts, proteins, endogenous serum peptides, and so forth.

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 24 N-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.

Apo-H (beta-2-glycoprotein) intact N-glycan analysis by MALDI-TOF-MS using sialic acid derivatization.

Apo-H is a plasma glycoprotein. Nearly 19% of the molecular weight of this protein is composed of glycans. Up- and down-regulation and structural changes in protein glycans provide diagnostic value for disease detection. Here, an efficient, sensitive, and optimized method is developed for Apo-H N-glycans analysis by MALDI-TOF-MS in positive mode. This bioanalytical method includes sample preparation, sample purification, and detection. An Apo-H enrichment method is developed using standard proteins by anti-Apo-H beads followed by enrichment from plasma samples. SDS-PAGE confirms the Apo-H protein enrichment, which is further verified by LC-MS/MS analysis. The lower ionization efficiency of sialylated glycan hampers their analysis by MALDI-MS. For this, stabilization of sialic acids is done by selective derivatization of carboxyl groups to differentiate between α(2,3)- and α(2,6)-linked sialic acids. Glycans are further purified by HILIC-SPE and analyzed by MALDI-MS. Several branched bi- and tri-antennary glycans with fucosylation and sialylation are identified. The reproducibility of the developed method is tested by analyzing multiple replicates of human plasma, where the same glycans are consistently identified. This method could be applied for the Apo-H glycan profiling of large clinical cohorts for diagnostic purposes.

Graphene oxide-metal oxide nanocomposites for on-target enrichment and analysis of phosphorylated biomolecules.

The surface of matrix-assisted laser desorption/ionization mass spectrometry target is modified for improved signal strength and detection of analytes. The developed method includes on-target enrichment and detection of phosphopeptides/phospholipids using graphene oxide-lanthanide metal oxides (samarium, gadolinium, dysprosium, and erbium) nanocomposites. Enriched phosphopeptides are detected using material enhanced laser desorption/ionization mass spectrometry and phospholipids by laser desorption/ionization-mass spectrometry. Nanocomposites are prepared using graphene oxide with respective metal salts at high pH. They are characterized for nano-morphology, chemistry, porosity, composition, crystallinity, and thermal stability. Phosphopeptides enrichment protocol is developed and optimized for tryptic ß-casein digest and that of phospholipids by phosphatidylcholine standard. Statistical analyses of phosphopeptides and phospholipids from milk show overlapping results for gadolinium, dysprosium, and erbium oxide nanocomposites. GO-Gd2 O3 has better enrichment efficiency and application as LDI material. Selectivity for GO-Dy2 O3 is 1:2500, for GO-Sm2 O3 is 1:3500, and 1:4000 for GO-Gd2 O3 . GO-Er2 O3 has a sensitivity of 25 fmol, whereas the highest sensitivity is down to 0.5 fmol for GO-Gd2 O3 . On-target enrichment is batch to batch reproducible with a standard deviation of <1, reduced time of enrichment to 10 min, and ease of operation compared to solid-phase batch extraction. The developed method enriches serum phosphopeptides characteristic of cancer-related phosphoproteins.

Iminodiacetic acid (IDA)-generated mesoporous nanopolymer: a template to relate surface area, hydrophilicity, and glycopeptides enrichment.

A three-step strategy is introduced to develop inherent iminodiacetic (IDA)-functionalized nanopolymer. SEM micrographs show homogenous spherical beads with a particle size of 500 nm. Further modification to COOH-functionalized 1,2-epoxy-5-hexene/DVB mesoporous nanopolymer enriches glycopeptides via hydrophilic interactions followed by their MS determination. Significantly high BET surface area 433.4336 m2 g-1 contributes to the improved surface hydrophilicity which is also shown by high concentration of ionizable carboxylic acids, 14.59 ± 0.25 mmol g-1. Measured surface area is the highest among DVB-based polymers and in general much higher in comparison to the previously reported BET surface areas of co-polymers, terpolymers, MOFs, and graphene-based composites. Thirty-one, 19, and 16 N-glycopeptides are enriched/identified by nanopolymer beads from tryptic digests of immunoglobulin G, horseradish peroxidase, and chicken avidin, respectively, without additional desalting steps. Material exhibits high selectivity (1:400 IgG:BSA), sensitivity (down to 0.1 fmol), regeneration ability up to three cycles, and batch-to-batch reproducibility (RSD > 1%). Furthermore, from 1 µL of digested human serum, 343 N-glycopeptide characteristics of 134 glycoproteins including 30 FDA-approved serum biomarkers are identified via nano-LC-MS/MS. The developed strategy to self-generate IDA on polymeric surface with improved surface area, porosity, and ordered morphology is insignia of its potential as chromatographic tool contributing to future developments in large-scale biomedical glycoproteomics studies.

Fabrication of Piperazine Functionalized Polymeric Monolithic Tip for Rapid Enrichment of Glycopeptides/Glycans.

Enrichment strategies are designed for the pretreatment of low-abundance glycans and glycopeptides prior to mass spectrometric (MS) analysis. Here, a tip-based strategy is being reported for the enrichment of glycopeptides and glycans using a piperazine modified polymeric monolithic tip. The tip is fabricated using the free radical polymerization. Fast separation (2 min) is achieved under optimized conditions with 20 cycles per step of loading, incubation, washing, and elution followed by MALDI-MS analysis. A total of 25, 22, and 34 glycopeptides covering all glycosylation sites are enriched by the modified tips from tryptic digests of horse radish peroxidase, chicken avidin, and human immunoglobulin G, respectively. Piperazine exhibits high selectivity 1:400 horse radish peroxidase/bovine serum albumin, sensitivity to 100 attomoles, recovery 89.51%, and batch to batch reproducibility (RSD > 1) in glycopeptides enrichment. Piperazine tips also enrich glycans from ovalbumin and human immunoglobulin G. High selectivity (1:1200, ovalbumin/BSA) and detection limit of 100 attomole is attained for glycans and furthermore 58 glycans are enriched from human serum. Thus, piperazine tips can be used as an enrichment tool for swift, cost-effective routine analysis of biological samples for separation of glycopeptides and glycans.

Boronic acid functionalized MOFs as HILIC material for N-linked glycopeptide enrichment.

Highly specific enrichment of N-linked glycopeptides from complex biological samples is crucial prior to mass spectrometric analysis. In this work, a hydrophilic metal-organic framework composite is prepared by the growth of UiO-66-NH2 on graphene sheets, followed by its post-synthetic modification to attach boronic acid to form GO@UiO-66-PBA. The fabrication of graphene oxide-MOF composite results in enhanced surface area with improved thermal and chemical stability. The synthesized MOF nanocomposite is characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and BET. A crystalline structure with high porosity offering large surface area and good hydrophilicity of the nanocomposite assists as an enrichment tool in glycoproteomics. The GO@UiO-66-PBA nanocomposite selectively enriches N-linked glycopeptides from tryptic digests of horseradish peroxidase (HRP) and immunoglobulin (IgG). GO@UiO-66-PBA nanoparticles show a low detection limit (1 fmol) and good specificity (1:200), reusability and reproducibility for N-linked glycopeptide enrichment from IgG digest. The binding capacity of GO@UiO-66-PBA is 84 mg/g for protein concentration, with a good recovery of 86.5%. A total of 372 N-linked glycopeptides corresponding to different glycoproteins are identified from only 1 µL of human serum digest. Thus, the presented research work can be an efficient separation platform for N-linked glycopeptide enrichment from complex samples, which can be extended to cost-effective routine analysis. Graphical abstract.

Boronic acid functionalized fibrous cellulose for the selective enrichment of glycopeptides.

Enrichment of glycoproteins has been important because of their dynamicity and role in biological systems. Study of glycoproteins is complex because of the simultaneous glycosylation and deglycosylation inside the body. Often employed affinities for glycopeptides are hydrazide, boronic acid, or physiosorbed lectin on support materials. Cellulose, a natural polysaccharide, has rich surface chemistry, stable structure, low cost and availability in different variants. In present study, fibrous cellulose is oxidized using periodate to modify with boronic acid. Attachment of boronic acid is confirmed by Fourier transform infrared spectroscopy. Particle size and morphology of boronic acid@fibrous cellulose is studied by scanning electron microscopy. The enrichment efficiency is evaluated by using horseradish peroxidase as model protein. Boronic acid@fibrous cellulose is selective up to 1:250 for spiked horseradish peroxidase in bovine serum albumin digest, sensitive down to 0.1 femtomol and recovering 88.15% glycopeptides. Moreover, protein binding capacity is determined as 213 mg/g and 41% sequence coverage of horseradish peroxidase protein with all eight glycosylation sites detected. Total of 18 glycopeptides are enriched from immunoglobulin digest showing ability of boronic acid@fibrous cellulose to enrich glycoproteins from multiglycoforms. Enrichment from human serum recovers 18% extracellular and 72% secreted glycoproteins via bottom-up approach and online tools.

Enrichment of HDL proteome and phospholipidome from human serum via IMAC/MOAC affinity.

High-density lipoproteins (HDLs) have anti-inflammatory and antioxidant properties and are potentially cardio-protective. Defective HDL function is caused by alterations in both the proteome and lipidome of HDL particles. As potential biomarkers, the development of analytical methods is necessary for the enrichment of HDLs. Therefore, a method for selective enrichment of HDLs using immobilized metal ion affinity chromatography (IMAC) and metal oxide affinity chromatography (MOAC) is presented. SPE-based isolation of HDLs from whole serum is adopted as an alternative to traditional ultracentrifugation methods followed by SDS-PAGE. The enrichment mechanism relies on isoelectric points of lipoproteins and metal oxide. Negatively charged lipoprotein particles interact with positively charged metal oxides and IMAC affinity, which acts as a cation. Identified proteins from HDL through MALDI-MS analysis are apo AI, AII, AIV, CI, CIII, E, J, M, H, serum amyloid A and other nonapoproteins that are part of HDL particles and perform cellular functions. This serum-based proteomics approach gives insight into the functional role of HDL. HDL-associated phospholipids have also been analyzed by LDI-MS. Results suggest that the adopted analytical strategy is a feasible idea to extract lipoproteins from serum. A comparative study of healthy and diseased samples using this approach will provide valuable information in future.

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 1 fmol µL-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 1 µL 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.

Metal-organic framework-based affinity materials in proteomics.

Metal-organic frameworks (MOFs) are an eminent addition to materials science research because of their versatile properties due to which their applications are wide spread in proteomics. They are used in various fields due to their characteristics like higher surface area, specific symmetry, ease of modification, and availability of a variety of ligands. As affinity sorbents, they have shown higher selectivity, sensitivity, and reproducibility than conventionally used materials. They are applied for the enrichment of phosphopeptides, glycopeptides, low mass peptides, and as laser desorption/ionization (LDI) matrices for small-molecule analysis. This review captures the insight of applying MOFs in the field of mass spectrometry-based proteomics. The specific features are discussed regarding MOFs as affinity sorbents for the selective capture of biological molecules like phosphopeptides and glycopeptides from complex samples. The potential of MOFs as LDI mass spectrometry (LDI-MS) matrices for small-molecule analysis is also evaluated. MOFs have also been used as enzymatic reactors for the digestion of proteins, prior to MS analysis. MOF-based affinity materials and bioreactors reduce proteome complexity and improve detection sensitivity and coverage. Size-exclusion effects of MOFs help in subtracting the abundant proteins in peptidomics. Several limitations of MOFs are addressed, which include stability under varying pH conditions, the unclear interaction mechanism between the MOFs and targeted analytes, and the non-specific binding that interferes during the analysis because of metal centers and ligands in the MOFs. This will open up MOF-based research to overcome the limitations and improve the performance of MOFs as selective and sensitive materials. Graphical abstract ᅟ.

Magnetite nanoparticles coated with chitosan and polyethylenimine as anion exchanger for sorptive enrichment of phosphopeptides.

An anion exchange solid-phase sorbent is described. Chitosan coated magnetite nanoparticles were modified with polyethylenimine which is positively charged at pH 3 and therefore can be used for the magnet-supported enrichment of phosphopeptides which are negatively charged at this pH value. A 2-step strategy was used to synthesize the sorbent. The materials were characterized by transmission electron microscopy, scanning electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction, thermogravimetry and magnetic moment analysis. The anion exchanger was applied to extract phosphopeptides from a ß-casein digest. Characteristic analytical figures include (a) a loading buffer of pH 3, (b) and elution buffer of pH 11, (c) a loading time of 5 min, (d) good selectivity (the ß-casein to BSA ratio is 1:1000), and (e) excellent sensitivity (1 fmol). The optimized method was applied to egg yolk digest, non-fat milk digest, and diluted human serum. Graphical abstractSchematic representation of synthesis of PEI@chitosan@Fe3O4 nanoparticles, and of the enrichment of phosphopeptides by magnetic solid phase extraction prior to the determination of the peptides by MALDI-MS analysis.

In-Tip Lanthanum Oxide Monolith for the Enrichment of Phosphorylated Biomolecules.

Polymeric monoliths fabricated in tips with embedded materials of choice are important in separation science. Polymeric backbone however interferes in the enrichment and thus affects efficiency. This work focuses on the in-tip fabrication of lanthanum oxide porous monolith and its application in the enrichment of phosphorylated peptides and lipids. Polycondensation reaction uses an aqueous solution of LaCl3·7H2O with N-methyl formamide as porogen and propylene oxide as initiator. The aging time of monolith and temperature condition for the reaction are optimized to attain porous monolithic tip. A comparison of (i) solid phase batch extraction using La2O3, (ii) La2O3 embedded in poly(glycidyl methacrylate (GMA)/divinylbenzene (DVB)) tip, and (iii) pure La2O3 monolithic tip shows improved enrichment efficiency in the case of pure La2O3 monolithic tip. The monolithic tip achieves selectivity of 1:4500 as compared to solid phase extraction (SPE)(1:3500) and limit of detection down to 0.25 fmol. The in-tip La2O3 monolith strategy has better batch to batch reproducibility, reduced time of enrichment, and ease of operation in comparison to solid phase batch extraction. The developed strategy enriches phospho- content from biological samples like phosvitin and lipovitellin from egg yolk and phospholipids/phosphopeptides from human serum. The enriched phospho- moieties are analyzed by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) except the phospholipids where laser desorption ionization (LDI)-MS is employed.

Hydrazide-functionalized affinity on conventional support materials for glycopeptide enrichment.

In affinity chromatography, enrichment of biomolecules is dependent on the selection of affinity sites immobilized onto a suitable support material. A few hydrazide - functionalized materials with surface modification protocols compatible to conventional support materials like silica and cellulose are reported. The study demonstrates the modification/derivatization pathways that can be adopted to modify the support materials with similar surface chemistry like cellulose, poly(GMA/DVB), or diamond. Poly(GMA/DVB) and cellulose represent hydrophilic supports whereas diamond is a hydrophobic support material. SEM images of three materials provide surface morphology whereas FT-IR confirms reaction completion and derivatization. These hydrazide - functionalized materials are applied to fetuin digest for glycopeptides enrichment and subsequently for selectivity and sensitivity assessment. Statistically, poly(GMA/DVB) shows 85.7% sensitivity with specificity of 88.8% in the enrichment experiments. Diamond offers hydrophobic interactions to non-glycopeptides and they co-elute with glycopeptides, resulting in reduced sensitivity down to 69.2%. Poly(GMA/DVB) shows recovery up to 89%, while recovery for cellulose and diamond is 83 and 71%, respectively. The materials enrich mono-N-linked-glycosylated peptide from tryptic digest of chicken avidin spiked in fetuin digest. The hydrazide group density on cellulose, poly(GMA/DVB), and diamond is 2.8, 2.3, and 2.1 mmol/g, respectively; this contributes towards the specificity and sensitivity of designed materials. The materials are also applied to serum samples and enriched glycopeptides characteristic of serum glycoproteins of clinical importance. Therefore this study provides routes for the economical surface modifications of support materials and to fabricate affinity materials with improved efficiency. Graphical Abstract Glycopeptides enrichment by hydrazine affinity.

Alumina nanocomposites: a comparative approach highlighting the improved characteristics of nanocomposites for phosphopeptides enrichment.

The work is based on the comparative study of metal oxide nanocomposites based on alumina in combination with two transition metal oxides (zirconia and titania) and two lanthanide oxides (ceria and lanthanum oxide). The choice is based on specific aims, i.e. to improve the limitations of individual metal oxides in phosphopeptide enrichment. The nanocomposites have shown improved phosphopeptide enrichment efficiency in comparison to the individual metal oxides. Alumina-zirconia show enhanced mono-phosphorylated peptide enrichment than ZrO2 whereas alumina-titania has better recovery of mono- and multi-phosphorylated peptides in comparison to individual TiO2. Alumina-ceria and alumina-lanthanum oxide overall enrich higher number of phosphopeptides. The alumina nanocomposites show higher selectivity and sensitivity for spiked ß-casein in BSA (1:1000) and diluted ß-casein digest (10 femtomole), respectively. Through the transition metal oxide nanocomposites, number of phosphoproteins from human serum are identified while this number is highest in case of alumina-lanthanum oxide nanocomposite. Thus the enrichment is affected by the choice of metal oxide in the nanocomposite based enrichment strategies.

Gadolinium oxide: Exclusive selectivity and sensitivity in the enrichment of phosphorylated biomolecules.

Selectivity and sensitivity define the dynamic applicability of separation and enrichment techniques. Owing to proteome complexity, numbers of separation media have been introduced in phosphoproteomics. Complex samples are pretreated to make the low-abundance molecules detectable by mass spectrometry. Gadolinium oxide nanoparticles, offering mono- and bi-dentate interactions, are optimized to capture the phosphopeptides. Selectivity of 1:11 000 is achieved for digested ß-casein phosphopeptides in bovine serum albumin digest background using gadolinium oxide nanoparticles. The limit of detection goes down to 1 attomole. With the optimized sample preparation protocol, gadolinium oxide nanoparticles enrich phosphopeptides of κ-casein (Ser148 and Ser170 ) from digested milk sample, fibrinogen alpha chain phosphopeptide (Ser609 ) along with four hydrolytic products of Ser22 -modified phosphopeptides from serum.

Advance workflow in lipoproteomics via polymeric ion exchanger.

A workflow is designed for the analysis of lipoproteins, high density lipoproteins (HDL), apoproteins, and lipid fraction, employing an organic polymeric anion exchanger through the enrichment of lipoproteins/peptides from serum. Polymeric separation media are chemically stable over the wide pH range. Poly(GMA/DVB), poly(GMA/EGDMA), and poly(GPE/DVB) are synthesized by radical polymerization, derivatized as strong anion exchangers, and used for lipoproteins enrichment. Lipoprotein's surface is covered by phospholipids, having phosphate groups, therefore lipoproteins are enriched by the interaction of anion exchanger with the phosphate groups and eluted at the pH of 7.5. HDL are further isolated by precipitating the very low-density lipoproteins (VLDL) and low-density lipoproteins (LDL) with phosphotungstic acid as precipitating reagent, followed by delipidation via liquid/liquid extraction. Apolipoproteins profiling is done by MALDI-MS, and lipids are analyzed using gold nanoparticles in the LDI-MS process. This study introduces a lipoproteomics work flow in separation science which analyses the intact lipoproteins. Furthermore, solid phase extraction (SPE)-based methodology is reported for the first time in lipoproteomics. Use of organic polymers, high reproducibility, detailed analysis of lipoproteins, apoproteins/peptides, and lipids from the single serum sample are the distinctive features of this workflow. Being biomarkers of numerous diseases, lipoproteins have clinical significance, and this workflow can be used at diagnostic and therapeutic levels.

Newly fabricated magnetic lanthanide oxides core-shell nanoparticles in phosphoproteomics.

Metal oxides show high selectivity and sensitivity toward mass spectrometry based enrichment strategies. Phosphopeptides/phosphoproteins enrichment from biological samples is cumbersome because of their low abundance. Phosphopeptides are of interest in enzymes and phosphorylation pathways which lead to the clinical links of a disease. Magnetic core-shell lanthanide oxide nanoparticles (Fe3O4@SiO2-La2O3 and Fe3O4@SiO2-Sm2O3) are fabricated, characterized by SEM, FTIR, and EDX and employed in the enrichment of phosphopeptides. The nanoparticles enrich phosphopeptides from casein variants, nonfat milk, egg yolk, human serum and HeLa cell extract. The materials and enrichment protocols are designed in a way that there are almost no nonspecific bindings. The selectivity is achieved up to 1:8500 using ß-casein/BSA mixture and sensitivity down to 1 atto-mole. Batch-to-batch reproducibility is high with the reuse of core-shell nanoparticles up to four cycles. The enrichment followed by MALDI-MS analyses is carried out for the identification of phosphopeptides from serum digest and HeLa cell extract. Characteristic phosphopeptides of phosphoproteins are identified from human serum after the enrichment, which have the diagnostic potential toward prostate cancer. Thus, the lanthanide based magnetic core-shell materials offer a highly selective and sensitive workflow in phosphoproteomics.

Development of new multifunctional terpolymer sorbent for proteomics applications.

Determination of the availability of phases for specific separations is an important task achieved by a separation chemist. This becomes vital when the complex samples like biofluids are dealt with in proteome science. The work presented here involves the synthesis and application of terpolymeric sorbent with different functionalizations adopted for the selective enrichment of biomolecules of interest from biological fluids. Synthesis of terpolymer was carried out by the radical polymerization of monomers: methyl acrylate, acrylic acid and vinyl acetate with diethylene glycol dimethacrylate as cross-linking agent, benzoyl peroxide as initiator and chloroform as a porogenic solvent. Characterization was done through Fourier transform infrared spectroscopy, scanning electron microscopy and nitrogen adsorption porosimetry. The polymer was further modified to immobilized metal ion affinity chromatographic material, with immobilized Fe(3+)/La(3+) ions that allowed phosphopeptide enrichment from tryptic digests of standard proteins as well as milk, egg yolk and human serum. Sensitivity of enrichment down to 50 fmol was achieved in the presence of complex protein background as bovine serum albumin. Hydrophobicity was introduced through octadecyl amine, which provides comparable results to ZipTip C18/C4 for desalting of complex mixtures of all caseins. Analysis of the enriched content was performed by Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

Au-nanomaterials as a superior choice for near-infrared photothermal therapy.

Photothermal therapy (PPT) is a platform to fight cancer by using multiplexed interactive plasmonic nanomaterials as probes in combination with the excellent therapeutic performance of near-infrared (NIR) light. With recent rapid developments in optics and nanotechnology, plasmonic materials have potential in cancer diagnosis and treatment, but there are some concerns regarding their clinical use. The primary concerns include the design of plasmonic nanomaterials which are taken up by the tissues, perform their function and then clear out from the body. Gold nanoparticles (Au NPs) can be developed in different morphologies and functionalized to assist the photothermal therapy in a way that they have clinical value. This review outlines the diverse Au morphologies, their distinctive characteristics, concerns and limitations to provide an idea of the requirements in the field of NIR-based therapeutics.