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We report herein a rationally designed pyrene linked substrate for quantitative protease activity assay via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). In this proof-of-concept study, a trypsin-specific peptide with the sequence of GGGGRG was selected to conjugate with pyrene forming a pyrene linked peptide probe, Py-GGGGRG. In the presence of trypsin, the Py-GGGGRG probe can be specifically hydrolyzed into Py-GGGGR. The introduction of pyrene greatly increased ionization efficiency of Py-peptides, and Py-peptides could be selectively captured from complex mixtures by a facially fabricated polystyrene coated MALDI plate through hydrophobic and π-π stacking interactions. As a result, trypsin activity can be directly quantified by relative intensity ratio of product and substrate via MALDI-TOF-MS without the use of external internal standard. A linear range of 0.1–10 μg/mL and a relatively low detection limit of 29 ng/mL were obtained. This method has also been successfully used for quantification of trypsin activity in urine and screening the inhibitors of trypsin. Besides, the proposed strategy was also validated for another protease, chymotrypsin, by using the probe Py-GGGGGGYG. Therefore, owing to simplicity, high-throughput capacity and quantificational accuracy, the proposed method shows great potential for activity assay of various proteases and screening their inhibitors via application of specific peptide sequences. fx1 • A rationally designed pyrene linked peptide probe was used for quantitative protease assay by MALDI-TOF-MS. • The incorporation of pyrene greatly increased the ionization efficiency of probe and enabled the on-line purification. • This proposed method was successfully used for quantification of trypsin assay in urine and screening its inhibitors. • This proposed method was simple, high-throughput and had great quantitative accuracy. • As a proof-of-concept study, this proposed approach also can be used for other protease assay. [ABSTRACT FROM AUTHOR]

Abstract We report a facile method for detection of metallothionein (MT), a promising clinically relevant biomarker, in spiked plasma samples. This method, for the first time, integrates molecularly imprinted polymers as purification/pretreatment step with matrix assisted laser desorption/ionization time-of-flight mass spectrometric detection and with laser ablation inductively coupled plasma mass spectrometry for analysis of MTs. The prepared MT-imprinted polydopamine layer showed high binding capacity and specific recognition properties toward the template. Optimal monomer (dopamine) concentration was found to be 16 mM of dopamine. This experimental setup allows to measure µM concentrations of MT that are present in blood as this can be used for clinical studies recognizing MT as marker of various diseases including tumour one. Presented approach not only provides fast sample throughput but also avoids the limitations of methods based on use of antibodies (e.g. high price, cross-reactivity, limited availability in some cases, etc.). Graphical abstract fx1 Highlights • Molecularly imprinted polymers as purification method for MT-1G was firstly used. • The conditions for the polymers preparation were optimized. • The purification step was then followed by MALDI-TOF mass spectrometry. • The concept was tested on the spiked blood serum. [ABSTRACT FROM AUTHOR]

This study aimed to assess the benefits of dithiothreitol (DTT)-based sample treatment for protein equalization to assess potential biomarkers for bladder cancer. The proteome of plasma samples of patients with bladder carcinoma, patients with lower urinary tract symptoms (LUTS) and healthy volunteers, was equalized with dithiothreitol (DTT) and compared. The equalized proteomes were interrogated using two-dimensional gel electrophoresis and matrix assisted laser desorption ionization time of flight mass spectrometry. Six proteins, namely serum albumin, gelsolin, fibrinogen gamma chain, Ig alpha-1 chain C region, Ig alpha-2 chain C region and haptoglobin, were found dysregulated in at least 70% of bladder cancer patients when compared with a pool of healthy individuals. One protein, serum albumin, was found overexpressed in 70% of the patients when the equalized proteome of the healthy pool was compared with the equalized proteome of the LUTS patients. The pathways modified by the proteins differentially expressed were analyzed using Cytoscape. The method here presented is fast, cheap, of easy application and it matches the analytical minimalism rules as outlined by Halls. Orthogonal validation was done using western-blot. Overall, DTT-based protein equalization is a promising methodology in bladder cancer research. [ABSTRACT FROM AUTHOR]

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has been proven as a useful and advanced technique in the identification of polymers and proteins. However, MALDI-TOF-MS still has the unavoidable drawback of self-signal interference with traditional organic matrices, which could suppress and overlap with the analyte signals in the low-mass region. In this work, MIL-101(Cr), a kind of metal-organic frameworks which possess high molecular weight, π-conjugated 3-D structure, coordinately unsaturated chromium sites (CUS) and strong absorption in the UV range, was employed to replace traditional organic matrices, and it was found that MIL-101(Cr) can dramatically eliminate the background peaks, showing high signal-to-noise level in the analysis of small molecules. As proof-of-concept, quercetin, daidzein, genistein and naringenin, members of flavonol family which widely exists in food and natural products, were successfully determined by utilizing MIL-101(Cr) as the surface-assisted matrix, and the detection of quercetin was sensitive with good salt tolerance and reproducibility. Under optimal conditions, the mass peak intensity exhibited good linear relationships in the range from 0.25 µg/mL-7.00 µg/mL for quercetin ( R 2 =0.996) with detection limit 2.11 ng/mL (3σ/k), making the identification of quercetin in sophora japonica successfully. With this strategy we have demonstrated the potentiality of MIL-101(Cr) nanomaterials as MALDI-MS matrix for the detection of small molecules. [ABSTRACT FROM AUTHOR]

Efficient and specific enrichment of low-abundant glycopeptides from complex biological samples is essential to glycoproteomics analysis. Herein, novel magnetic zwitterionic hydrophilic nanoparticles based on thiol-ene click chemistry were synthesized. The functionalized magnetic nanoparticles exhibited superior performance in glycopeptide enrichment of HRP tryptic digest, demonstrating low detection limit (0.04 ng/μL), high selectivity (a mixture of HRP and BSA at the mass ratio of 1:50) and reproducibility (5 repeating cycles). In addition, the material was successfully applied to glycopeptide enrichment from human serum. The outstanding results indicate the potential of the method in the development of glycoproteomics analysis in real biological samples. [ABSTRACT FROM AUTHOR]

Peptides ligands with high affinity and high specificity towards specific targets is catching a good deal of interests in biomedical field. Traditional peptide screening procedure involves selection, sequencing and characterization and each step is time-consuming and labor-intensive. The combination between different analytical methods could provide an integrated plan for efficient peptide screening. We report herein a label-free detection microarray system to facilitate the whole one-bead-one-compound (OBOC) peptide screening process. A microwell array chip with two identical units can trap the candidate peptide beads in one-well-one-bead manner. Peptides on beads were photo-released in situ in the well and partly transferred to two identical chips for Surface Plasmon Resonance imaging (SPRi), and peptide left in the bi-unit microwell array chip was remain for in situ single bead sequencing by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). Using the bi-unit imprinted chip system, affinity peptides towards AD protein were efficiently screened out both qualitatively and quantitatively from 10 4 candidates. The method provides a universal solution for high efficiency and high throughput ligands screening. [ABSTRACT FROM AUTHOR]

Abstract: To make the use of MALDI-based mass spectrometry feasible for the fast analysis of oxosteroids, three new aromatic probes have been designed to be used simultaneously as derivatisation agents and MALDI matrices. This concept brings a number of benefits: the sample handling is reduced, the workflow is less time consuming allowing high throughput and the interferences caused by the MALDI matrix are avoided. Identification was successfully attained for all oxosteroids used in this study. As proof-of-concept, the identification of oxosteroids in urine was performed to evaluate the robustness of the new methodology. The oxosteroids 17-α-methyltestosterone, nandrolone, boldenone, 17-α-Trenbolone, fluoxymesterolone, mesterolone and bolasterone were identified in human urine at the minimum concentration level recommended by the world anti-doping agency, 2ng/mL. [Copyright &y& Elsevier]

Abstract: Determination of perfluorinated compounds (PFCs) is very important because of their potential hazards to the environment and human health. In present work, 1,8-bis (tetramethylguanidino)-naphthalene (TMGN), a superbasic proton sponge, was firstly employed as the matrix for quantitative detection of acidic PFCs in environmental water samples by Matrix-assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF-MS). Several acidic PFCs, such as perfluorooctanesulfonate (PFOS) and perfluorooctanoic acid (PFOA), were selected as model analytes for demonstrating the feasibility of the detection method. The results showed that deprotonated ions of these PFCs were detected without any other matrix ions interference. The achieved sensitivity with TMGN for PFOS detection was ten-fold higher than that with 1,8-bis (dimethyl-amino)-naphthalene (DMAN) which was used for the detection of fatty acid by MALDI-TOF-MS. The high sensitivity of this method made it feasible to monitor and quantify acidic PFCs in complicated environmental water samples. Furthermore, a novel combined strategy of solid phase extraction (SPE) followed by MALDI-TOF-MS detection was developed for quantifying PFCs in environmental water samples. The calibration curves with a wide linear dynamic range (0.1–10ngL−1 for PFOS, PFHxS, and PFBS, and 0.5–50ngL−1 for PFOA, PFNA, and PFDA.) were obtained. The limit of detection (LOD) for PFOS of this method was 0.015ngL−1 (a signal-to-noise ratio of 3), which was lower than the LOD (0.036ngL−1) obtained by high-pressure liquid chromatography/tandem mass spectrometry (LC–MS/MS) method. Moreover, the strategy was used to detect the selected PFCs in water samples collected from Xiaoqinghe river and Gaobeidian wastewater. The achieved concentrations of PFCs were closed to those obtained by LC–MS/MS method. It is indicated that the proposed MALDI-TOF-MS method with TMGN as the matrix is much reliable and can be used as an alternative method to detect trace PFCs in environmental water samples. [Copyright &y& Elsevier]

Abstract: We report the first use of functionalized Ag2Se nanoparticles (NPs) as effective extracting probes for NPs-based liquid-phase microextraction (NPs-LPME) to analyze hydrophobic peptides and proteins from biological samples (urine and plasma) and soybean in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Surface modified functional groups such as octadecanethiol (ODT) and 11-mercaptoundecanoic acid (MUA) on Ag2Se NPs were found to play an important role for efficient extraction of peptides and proteins from test samples through hydrophobic interactions. The peptides can be efficiently extracted using functionalized Ag2Se NPs as extracting probes in the presence of high concentration of matrix interferences such as 4M urea, 0.5% Triton X-100 and 3% NaCl. Ag2Se@ODT NPs have shown better extraction efficiency and detection sensitivity for peptides than Ag2Se@MUA NPs, bare Ag2Se NPs and conventional MALDI-MS. The LODs are 20–68nM for valinomycin and 100–180nM for gramicidin D using Ag2Se@ODT NPs-LPME in the MALDI-MS. The current approach is highly sensitive and the target analytes can be effectively isolated without sample loss and efficiently analyzed in MALDI-MS. [ABSTRACT FROM AUTHOR]

Abstract: Four strains of Listeria monocytogenes with different levels of virulence were studied. Two strains were consistently evaluated as virulent (strain 3077) and of low virulence (strain 3993), whereas the other two strains (3006 and 3049) originated conflicting results in what the evaluation tests were concerned: both were shown to exhibit low virulence when evaluated by in vitro assays, but virulent when the analyses were performed under in vivo conditions. To clarify the virulence potential of the selected strains, a proteomic approach was used after incubating L. monocytogenes cultures under conditions favoring the expression of virulence factors (minimal medium, at 37°C). Bacterial proteins present in the liquid culture media were precipitated from late exponential phase cultures, fractionated by SDS-PAGE and identified by MALDI-TOF-MS. Three virulence factors differentially expressed were detected: protein p60, listeriolysin O (LLO) and internalin C (InlC). Clustering analysis of the four L. monocytogenes strains based on their secretome profiles allowed their categorization in two groups: the virulent group, composed by strains 3077 and 3049, and the low virulence group, containing strains 3993 and 3006. The results presented in this work suggest that the virulent potential of a particular L. monocytogenes strain may be predicted from the levels of both listeriolysin O (LLO) and internalin C (InlC) present in its secretome when the bacterium is grown under conditions favoring the expression of virulence factors. Following validation of this proposal through the analysis of a large array of strains, this methodology exhibits a great potential to be developed into an accurate and rapid method to characterize L. monocytogenes strain virulence. [ABSTRACT FROM AUTHOR]

Abstract: CdS quantum dots (∼5nm) are used as multifunctional nanoprobes as an effective matrix for large proteins, peptides and as affinity probes for the enrichment of tryptic digest proteins (lysozyme, myoglobin and cytochrome c) in laser desorption/ionization time-of-flight mass spectrometry (LDI-TOF MS). The use of CdS quantum dots (CdS QDs) as the matrix allows acquisition of high resolution LDI mass spectra for large proteins (5000–80,000Da). The enhancement of mass resolution is especially notable for large proteins such as BSA, HSA and transferrin (34–49 times) when compared with those obtained by using SA as the matrix. This technique demonstrates the potentiality of LDI-TOF-MS as an appropriate analytical tool for the analysis of high-molecular-weight biomolecules with high mass resolution. In addition, CdS QDs are also used as matrices for background-free detection of small biomolecules (peptides) and as affinity probes for the enrichment of tryptic digest proteins in LDI-TOF-MS. [ABSTRACT FROM AUTHOR]

Abstract: The decision peptide-driven tool implements a software application for assisting the user in a protocol for accurate protein quantification based on the following steps: (1) protein separation through gel electrophoresis; (2) in-gel protein digestion; (3) direct and inverse 18O-labeling and (4) matrix assisted laser desorption ionization time of flight mass spectrometry, MALDI analysis. The DPD software compares the MALDI results of the direct and inverse 18O-labeling experiments and quickly identifies those peptides with paralleled loses in different sets of a typical proteomic workflow. Those peptides are used for subsequent accurate protein quantification. The interpretation of the MALDI data from direct and inverse labeling experiments is time-consuming requiring a significant amount of time to do all comparisons manually. The DPD software shortens and simplifies the searching of the peptides that must be used for quantification from a week to just some minutes. To do so, it takes as input several MALDI spectra and aids the researcher in an automatic mode (i) to compare data from direct and inverse 18O-labeling experiments, calculating the corresponding ratios to determine those peptides with paralleled losses throughout different sets of experiments; and (ii) allow to use those peptides as internal standards for subsequent accurate protein quantification using 18O-labeling. In this work the DPD software is presented and explained with the quantification of protein carbonic anhydrase. [ABSTRACT FROM AUTHOR]

Abstract: We report in this work a fast protocol for protein quantification and for peptide mass mapping that rely on 18O isotopic labeling through the decoupling procedure. It is demonstrated that the purity and source of trypsin do not compromise the labeling degree and efficiency of the decoupled labeling reaction, and that the pH of the labeling reaction is a critical factor to obtain a significant 18O double labeling. We also show that the same calibration curve can be used for MALDI protein quantification during several days maintaining a reasonable accuracy, thus simplifying the handling of the quantification process. In addition we demonstrate that 18O isotopic labeling through the decoupling procedure can be successfully used to elaborate peptide mass maps. BSA was successfully quantified using the same calibration curve in different days and plasma from a freshwater fish, Cyprinus carpio, was used to elaborate the peptide mass maps. [Copyright &y& Elsevier]

Abstract: Promising profiling techniques based on new material/solid phase extraction for capturing “molecular signatures” from body fluids are being coupled to MALDI-TOF-MS. Sample preparation significantly influences spectrum quality in this ionization method. Mesoporous silica beads (MSB), by the means of nano-sized porous channels with high surface area, enable harvesting of peptides from plasma and serum excluding large size proteins. We have investigated the morphology of a sample slurry, developed as a new tool for plasma peptides enrichment based on mesoporous materials. Our study highlights a correlation between crystals morphology and enhanced performances in MALDI-TOF-MS analysis. This is the first report which correlates the increase in signal intensity with crystal formation in samples preparations which make use of various kinds of slurries for the analysis of samples clinically relevant like human plasma. [Copyright &y& Elsevier]

Abstract: Molecular mass spectrometry (MS) analysis of protein phosphorylation is partially limited by the molecular specie specificity of the analytical responses that might impair both qualitative and quantitative performances. Elemental MS, such as inductively coupled plasma mass spectrometry (ICP-MS) can overcome these drawbacks; in fact, analytical performance is theoretically independent of the molecular structure of a target analyte naturally containing the elements of interest. Nevertheless, isobaric interferences derived from sample matrix and laboratory environment can hinder the quantitative determination of both phosphorus (P) and sulfur (S) as 31P+ and 32S+ by inductively coupled plasma quadrupole mass spectrometry (ICP-QMS) under standard plasma conditions. These interferences may be overcome by quantifying P and S as oxide ions 31P16O+ and 32S16O+, respectively. In this study, we present a systematic investigation on the effect of plasma instrumental conditions on the oxide ion responses by a design of experiment approach for the simultaneous ICP-QMS determination of P and S (31P16O+ and 32S16O+, respectively) in protein samples without the use of dynamic reaction, collision reaction cells or pre-addition of oxygen as reactant gas in the torch. The proposed method was evaluated in terms of limit of detection, limit of quantification, linearity, repeatability, and trueness. Moreover, detection and quantification capabilities of the optimized method were compared to the standard plasma mode for determination of 31P+ and 34S+. Spectral and non-spectral interferences affecting the quantification of 31P+, 31P16O+ and 32S16O+ were also studied. The suitability of inorganic elemental standards for P and S quantification in proteins was assessed. The method was applied to quantify the phosphorylation stoichiometry of commercially available caseins (bovine β-casein, native and dephosphorylated α-casein) and results were confirmed by Matrix Assisted Laser Desorption Ionization Time of Flight MS analysis. We demonstrate that ICP-QMS, by quantifying P and S as oxide ions, was able to accurately calculate the degree of phosphorylation of β-casein and α-casein and to detect specific partial enzymatic dephosphorylation. The collected results might lead to further development of ICP-QMS interfaces optimized for protein phosphorylation studies and for proteomics investigations. [Copyright &y& Elsevier]

Abstract: A new clean fast (8min) method for in-solution protein digestion without detergent or urea for protein identification by peptide mass fingerprint and mass spectrometry-based techniques is proposed. The new method avoids the use of time consuming desalting procedures entailing the following four steps done under the effect of an ultrasonic field provided by a sonoreactor: denaturation (1min) in a mixed solution of water:acetonitrile 1/1 (v/v); protein reduction (1min); protein alkylation (1min); and protein digestion (5min). Five proteins with masses comprised between 14.4kDa and 97kDa and the protein split-soret cytochrome c from D. desulfuricans ATCC27774, were successfully identified with this procedure. No differences were found in the sequence coverage or in the number of peptides matched when the new clean method was compared to another one using urea. Twofold better signal-to-noise ratios were obtained in the MALDI spectra from protein samples prepared with the new method when comparing it with a method using urea. The new digestion method avoids the need to remove salt content and increases throughput (six samples at once) while reducing sample loss and contamination from sample handling. [Copyright &y& Elsevier]

Abstract: Different ultrasonic devices including ultrasonic bath with dual frequency, sonoreactor and ultrasonic probe, were tested for their viability in the sample treatment for polymer characterization by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis. The effect of sonication frequency (35kHz, 40kHz and 130kHz), sonication amplitude, and sonication time on the polymer''s number-average molecular weight (M n) and weight-average molecular weight (M w) were investigated. The effect of those variables in the molecular mass distribution of three polymer standards, poly(styrene) 2000Da and 10,000Da and poly(ethylene glycol) 1000Da, was evaluated. In addition, the influence of ultrasonic energy on the sample treatment as a function of the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI) matrix was also studied through two common standard matrices, dithranol and 2,5-dihydroxybenzoic acid. The results obtained show that the ultrasonic bath at 35kHz is the best option for the purpose of fast sample treatment for polymer characterization. The M n and M w values obtained for this ultrasonic device and for the three polymers tested using dithranol as MALDI matrix, were not statistically different from the ones acquired with vortex mixing and also were in concordance with the values recommended by the polymer manufacturers. [Copyright &y& Elsevier]

Abstract: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) have become established as very efficient and sensitive biopolymer and elemental mass spectrometric techniques for studying metal-binding proteins (metalloproteins) in life sciences. Protein complexes present in rat tissues (liver and kidney) were separated in their native state in the first dimension by blue native gel electrophoresis (BN-PAGE). Essential and toxic metals, such as zinc, copper, iron, nickel, chromium, cadmium and lead, were detected by scanning the gel bands using quadrupole LA-ICP-MS with and without collision cell as a microanalytical technique. Several proteins were identified by using MALDI-TOF-MS together with a database search. For example, on one protein band cut from the BN-PAGE gel and digested with the enzyme trypsin, two different proteins – protein FAM44B and cathepsin B precursor – were identified. By combining biomolecular and elemental mass spectrometry, it was possible to characterize and identify selected metal-binding rat liver and kidney tissue proteins. [Copyright &y& Elsevier]

Proteinase K (ProK) is used for the degradation of proteins in cell lysates to isolate nucleic acids, and for the speciation of proteins for mass spectrometry analysis. In this work, a novel and sensitive immobilization process was developed for examination of protein mixtures by combining MALDI-ToF-MS and nLC-TIMS-ToF-MS/MS systems. To achieve these goals, magnetic nanoparticles (MPs) were prepared via thermal coprecipitation reaction under alkaline condition. The MPs were grafted with a silica layer (i.e., 3-(2,3-epoxypropoxy) propyltrimethoxysilane; EPTES) containing reactive epoxy groups. Then, the silica-grafted magnetic particles were coated with a long chain hydrophilic poly(ethylene glycol) diamine polymer (PEGDAP). The prepared materials were characterized by the Brunauer–Emmett–Teller (BET) method, X-ray diffraction (XRD), scanning electron microscopy (SEM) and attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. The VSM data show that the MPs@EPTES@PEGDAP has paramagnetic performance with a saturation magnetization of approximately 32.3 emu g−1. Proteinase K (EC 3.4.21.64) was covalently immobilized on the MPs@EPTES by reaction of its epoxy groups with amine groups of the enzyme. On the other hand, the ProK was immobilized on the MPs@EPTES@PEGDAP after activation with glutaraldehyde and the immobilization reaction was realized by the coupling reaction between aldehyde groups of the support and amine groups of the enzyme. The amounts of immobilized ProK on the MPs@EPTES and MPs@EPTES@PEGDAP were found to be 27.4 and 19.6 mg g−1and the retained activities were determined to be 29 and 87%, respectively. For the first time, some important features such as thermal and storage stabilities, reusability and potential use in protein speciation for mass spectrometry-based techniques were also evaluated. For examples, after six weeks of storage at 4 °C, the immobilized ProK on the MPs@EPTES@PEGDAP-ProK still maintained 59% of its initial activity. However, at the end of the six-week storage period, its free counterpart had lost all of its initial activity. The immobilized ProK was also utilized for degradation and identification of model proteins (i.e., α-2-HS glycoprotein, β-casein, bovine serum albumin and immunoglobulin). After enzymatic treatment, the digested peptides were analyzed and mapped by using nLC-TIMS-ToF-MS/MS systems. Image 1 • Proteinase K immobilized silica coated MP's were used for digestion of different proteins. • Immobilized proteinase K showed good digestion efficiency, thermal and storage stabilities. • Immobilized enzyme provided an ultrafast peptide synthesis for label-free speciation of proteins. • The same immobilized enzyme was used many times for degradation of various proteins. • The digested peptides were analyzed and mapped using MALDI-ToF-MS and nLC-TIMS-ToF-MS/MS systems. [ABSTRACT FROM AUTHOR]

Body fluid N -glycome analysis as well as glyco-proteoform profiling of existing protein biomarkers potentially provides a stratification layer additional to quantitative, diagnostic protein levels. For clinical omics applications, the collection of a dried blood spot (DBS) is increasingly pursued as an alternative to sampling milliliters of peripheral blood. Here we evaluate DBS cards as a blood collection strategy for protein N -glycosylation analysis aiming for high-throughput clinical applications. A protocol for facile N -glycosylation profiling from DBS is developed that includes sialic acid linkage differentiation. This protocol is based on a previously established total plasma N -glycome mass spectrometry (MS) method, with adjustments for the analysis of DBS specimens. After DBS-punching and protein solubilization N -glycans are released, followed by chemical derivatization of sialic acids and MS-measurement of N -glycan profiles. With this method, more than 80 different glycan structures are identified from a DBS, with RSDs below 10% for the ten most abundant glycans. N -glycan profiles of finger-tip blood and venous blood are compared and short-term stability of DBS is demonstrated. This method for fast N -glycosylation profiling of DBS provides a minimally invasive alternative to conventional serum and plasma protein N -glycosylation workflows. With simplified blood sampling this DBS approach has vast potential for clinical glycomics applications. Image 1 • A method was developed for N-glycome profile analysis from dried blood spots. • The method: sampling, glycan release, derivatization, purification and MS-analysis. • Short term stability shows great potential for storage of dried blood spots. [ABSTRACT FROM AUTHOR]

A novel enzymatic reactor was prepared by incorporating SBA-15 nanoparticles into hybrid organic-inorganic monolith and immobilizing trypsin with glutaraldehyde as bridging reagent. Preparation and operation conditions including nanoparticles percentage and residence time were optimized to improve the digestion efficiency. The digestion products were characterized by Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) with sequence coverage of 50%, 93% and 71% for bovine serum albumin, myoglobin and cytochrome C, while consuming only about 19s in dynamic mode. Compared with enzymatic reactor without nanoparticles incorporated, the enzymatic reactor with SBA-15 nanoparticles embedded achieved higher digestion efficiency by introducing more trypsin, which was originated from combination of SBA-15 nanoparticles and hybrid organic-inorganic monolith., (© 2013 Elsevier B.V. All rights reserved.)