Rapid fingerprinting of a highly glycosylated fusion protein by microfluidic chip-based capillary electrophoresis-mass spectrometry.

Protein glycosylation can impact the efficacy, safety, and pharmacokinetics of therapeutic proteins. Achieving uniform and consistent protein glycosylation is an important requirement for product quality control at all stages of therapeutic protein drug discovery and development. The development of a new microfluidic CE device compatible with MS offers a fast and sensitive orthogonal mode of high-resolution separation with MS characterization. Here, we describe a fast and robust chip-based CE-MS method for intact glycosylation fingerprinting of a therapeutic fusion protein with complex sialylated N and O-linked glycoforms. The method effectively separates multiple sialylated glycoforms and offers a rapid detection of changes in glycosylation profile in 6 min.

Thiol-ene Monolithic Pepsin Microreactor with a 3D-Printed Interface for Efficient UPLC-MS Peptide Mapping Analyses.

To improve the sample handling, and reduce cost and preparation time, of peptide mapping LC-MS workflows in protein analytical research, we here investigate the possibility of replacing conventional enzymatic digestion methods with a polymer microfluidic chip based enzyme reactor. Off-stoichiometric thiol-ene is utilized as both bulk material and as a monolithic stationary phase for immobilization of the proteolytic enzyme pepsin. The digestion efficiency of the, thiol-ene based, immobilized enzyme reactor (IMER) is compared to that of a conventional, agarose packed bed, pepsin IMER column commonly used in LC-MS based protein analyses. The chip IMER is found to rival the conventional column in terms of digestion efficiency at comparable residence time and, using a 3D-printed interface, be directly interfaceable with LC-MS.

Development of an enzymatic reactor applying spontaneously adsorbed trypsin on the surface of a PDMS microfluidic device.

Herein, a microfluidic device (MD) containing immobilized trypsin for rapid and efficient proteolysis was described. Trypsin was immobilized via non-specific protein adsorption onto the hydrophobic poly(dimethylsiloxane) (PDMS) channel wall of the MD. Peptide mapping of bovine serum albumin (BSA) samples was carried out to estimate the stability of trypsin adsorbed on PDMS surface. Peptide maps of BSA samples were obtained by capillary zone electrophoresis (CZE), the RSD% for migration times were under 1%. Several proteins (hemoglobin, myoglobin, lysozyme, and BSA) in a wide molecular size range (15-70 kDa) were digested efficiently with ∼50 s contact time. The number of separated peaks correlated well with the expected number of peptides formed in the complete tryptic digestion of the proteins. Peptide mass fingerprinting of BSA and human serum was carried out. Trypsin retained its activity for 2 h; within this period, the MD can be used for multiple digestions. The main properties of this device are simple channel pattern, simple immobilization procedure, regenerability, and disposability; all these features make this MD one of the simplest yet applicable enzymatic microreactors. Graphical abstract Development of microfluidic device including a serpentine channel as an enzyme reactor for protein digestion.

Exploration of doubtful cases of leucine and isoleucine discrimination in mass spectrometric peptide sequencing by electron-transfer and higher-energy collision dissociation-based method.

Electron-transfer dissociation (ETD) and electron-transfer and higher-energy collision dissociation (EThcD) spectra of short tryptic peptides with leucine/isoleucine residues in neighboring positions demonstrate intensive w-ions. On the contrary, u-ions possess very low intensities (if present at all). Therefore radical site migration is negligible in the applied conditions while ETD (EThcD) spectra allow for the reliable discrimination of the isomeric residues in the sequencing process. The presence of a fragment ion 43.055 mass units lower than z2-ion of peptides with IK sequence at their C-termini was shown to be a result of alternative fragmentation starting from the loss of propylammonium ion from the doubly protonated peptide molecule and formation of an oxazole fragment ion.

Reversed phase liquid chromatography hyphenated to continuous flow-extractive desorption electrospray ionization-mass spectrometry for analysis and charge state manipulation of undigested proteins.

The application of continuous flow-extractive desorption electrospray ionization (CF-EDESI), an ambient ionization source demonstrated previously for use with intact protein analysis, is expanded here for the coupling of reversed phase protein separations to mass spectrometry. This configuration allows the introduction of charging additives to enhance detection without affecting the chromatographic separation mechanism. Two demonstrations of the advantages of CF-EDESI are presented in this work. First, a proof-of- principle is presented to demonstrate the applicability of hyphenation of liquid chromatography (LC) to CF- EDESI. LC-CF-EDESI-MS has good sensitivity compared to LC-electrospray ionization (ESI)-mass spectrometry. Second, the supercharging mechanism investigated in CF-EDESI provides an insight into a highly debated supercharging process in ESI. The results indicate that the mechanism of protein charging seen in HPLC-CF-EDESI is different from supercharging phenomena in conventional ESI. The surface tension mechanism and binding mechanism may both contribute to protein supercharging in ESI.

A novel platform for automated high-throughput fluxome profiling of metabolic variants.

Advances in metabolic engineering are enabling the creation of a large number of cell factories. However, high-throughput platforms do not yet exist for rapidly analyzing the metabolic network of the engineered cells. To fill the gap, we developed an integrated solution for fluxome profiling of large sets of biological systems and conditions. This platform combines a robotic system for (13)C-labelling experiments and sampling of labelled material with NMR-based isotopic fingerprinting and automated data interpretation. As a proof-of-concept, this workflow was applied to discriminate between Escherichia coli mutants with gradual expression of the glucose-6-phosphate dehydrogenase. Metabolic variants were clearly discriminated while pathways that support metabolic flexibility towards modulation of a single enzyme were elucidating. By directly connecting the data flow between cell cultivation and flux quantification, considerable advances in throughput, robustness, release of resources and screening capacity were achieved. This will undoubtedly facilitate the development of efficient cell factories.

Analysis of monoclonal antibody by a novel CE-UV/MALDI-MS interface.

mAbs are highly complex proteins that present a wide range of microheterogeneity that requires multiple analytical methods for full structure assessment and quality control. As a consequence, the characterization of mAbs on different levels is particularly product- and time-consuming. CE-MS couplings, especially to MALDI, appear really attractive methods for the characterization of biological samples. In this work, we report the last instrumental development and performance of the first totally automated off-line CE-UV/MALDI-MS/MS. This interface is based on the removal of the original UV cell of the CE apparatus, modification of the spotting device geometry, and creation of an integrated delivery matrix system. The performance of the method was evaluated with separation of five intact proteins and a tryptic digest mixture of nine proteins. Intact protein application shows the acquisition of electropherograms with high resolution and high repeatability. In the peptide mapping approach, a total number of 154 unique identified peptides were characterized using MS/MS spectra corresponding to average sequence coverage of 64.1%. Comparison with NanoLC/MALDI-MS/MS showed complementarity at the peptide level with an increase of 42% when using CE/MALDI-MS coupling. Finally, this work represents the first analysis of intact mAb charge variants by CZE using an MS detection. Moreover, using a peptide mapping approach CE-UV/MALDI-MS/MS fragmentation allowed 100% sequence coverage of the light chain and 92% of the heavy chain, and the separation of four major glycosylated peptides and their structural characterization.

A silicon nanomembrane detector for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) of large proteins.

We describe a MALDI-TOF ion detector based on freestanding silicon nanomembrane technology. The detector is tested in a commercial MALDI-TOF mass spectrometer with equimolar mixtures of proteins. The operating principle of the nanomembrane detector is based on phonon-assisted field emission from these silicon nanomembranes, in which impinging ion packets excite electrons in the nanomembrane to higher energy states. Thereby the electrons can overcome the vacuum barrier and escape from the surface of the nanomembrane via field emission. Ion detection is demonstrated of apomyoglobin (16,952 Da), aldolase (39,212 Da), bovine serum albumin (66,430 Da), and their equimolar mixtures. In addition to the three intact ions, a large number of fragment ions are also revealed by the silicon nanomembrane detector, which are not observable with conventional detectors.

Rapid glycopeptide enrichment and N-glycosylation site mapping strategies based on amine-functionalized magnetic nanoparticles.

Glycoproteins secreted or expressed on the cell surface at specific pathophysiological stages are well-recognized disease biomarkers and therapeutic targets. While mapping of specific glycan structures can be performed at the level of released glycans, site-specific glycosylation and identification of specific protein carriers can only be determined by analysis of glycopeptides. A key enabling step in mass spectrometry (MS)-based glycoproteomics is the ability to selectively or non-selectively enrich for the glycopeptides from a total pool of a digested proteome for MS analysis since the highly heterogeneous glycopeptides are usually present at low abundance and ionize poorly compared with non-glycosylated peptides. Among the most common approaches for non-destructive and non-glycan-selective glycopeptide enrichment are strategies based on various forms of hydrophilic interaction liquid chromatography (HILIC). We present here a variation of this method using amine-derivatized Fe(3)O(4) nanoparticles, in concert with in situ peptide N-glycosidase F digestion for direct matrix-assisted laser desorption/ionization­mass spectrometry analysis of N-glycosylation sites and the released glycans. Conditions were also optimized for efficient elution of the enriched glycopeptides from the nanoparticles for on-line nanoflow liquid chromatography­MS/MS analysis. Successful applications to single glycoproteins as well as total proteomic mixtures derived from biological fluids established the unrivaled practical versatility of this method, with enrichment efficiency comparable to other HILIC-based methods.

Polydopamine-assisted immobilization of trypsin onto monolithic structures for protein digestion.

Inspired by the catechol-rich adhesive proteins of the mussel foot, we report a simple and versatile aqueous approach for the immobilization of trypsin onto silica and titania monolithic supports. The method involves in-situ coating of the monolithic substrates with a catechol-containing biomimetic polymer (polydopamine) derived from the polymerization of dopamine under alkaline pH, followed by conjugation of trypsin to the polydopamine polymer coating. The trypsin immobilization efficiency onto the monolithic materials was investigated as a function of different preparation parameters such as dopamine concentration and coating time. The enzymatic activity of the immobilized trypsin reactors was evaluated, and mass spectrometry based proteomic analysis was demonstrated by digestion of a model protein. The method presented in this manuscript has broad potential for immobilization of trypsin and other enzymes onto a wide variety of monolithic supports, due to the ability of polydopamine to act as a primer for covalent immobilization of proteins.

Mapping O-GlcNAc modification sites on tau and generation of a site-specific O-GlcNAc tau antibody.

The microtubule-associated protein tau is known to be post-translationally modified by the addition of N-acetyl-D: -glucosamine monosaccharides to certain serine and threonine residues. These O-GlcNAc modification sites on tau have been challenging to identify due to the inherent complexity of tau from mammalian brains and the fact that the O-GlcNAc modification typically has substoichiometric occupancy. Here, we describe a method for the production of recombinant O-GlcNAc modified tau and, using this tau, we have mapped sites of O-GlcNAc on tau at Thr-123 and Ser-400 using mass spectrometry. We have also detected the presence of a third O-GlcNAc site on either Ser-409, Ser-412, or Ser-413. Using this information we have raised a rabbit polyclonal IgG antibody (3925) that detects tau O-GlcNAc modified at Ser-400. Further, using this antibody we have detected the Ser-400 tau O-GlcNAc modification in rat brain, which confirms the validity of this in vitro mapping approach. The identification of these O-GlcNAc sites on tau and this antibody will enable both in vivo and in vitro experiments designed to understand the possible functional roles of O-GlcNAc on tau.

Combination of acid labile detergent and C18 Empore™ disks for improved identification and sequence coverage of in-gel digested proteins.

A protocol for improved extraction of peptides from in-gel protein digests, using a combination of the acid labile surfactant, sodium deoxycholate (SDC) and C18 Empore™ membranes, is presented. This approach results in better mass spectrum quality, higher numbers of identified peptide peaks and improved identification scores compared to standard tryptic digestion protocols, or protocols using only SDC or only C18 Empore™ disks. The advantages of the new protocol are demonstrated for two different types of samples: Merino wool intermediate filament proteins and Elaeis guineensis (oil palm) mesocarp proteins.

Improved instrumentation for large-size two-dimensional protein maps.

Novel instrumentation for performing large-size (>25 cm) 2-D maps is reported here. To perform the first dimension, we developed a power supply that can deliver a voltage of up to 15,000 V and allows regulation of current (up to 200 µA) onto each individual focusing IPG strip. The IEF strip tray can accommodate up to 12 IPG strips and the electrodes slide on a ruler, thus permitting running strips of any length up to 45 cm. In addition, this apparatus also includes a second power supply that allows the performance of electrophoresis at high amperage (400 mA) and a Peltier system that allows a 10-80°C temperature control.

Rapid and efficient glycoprotein identification through microwave-assisted enzymatic digestion.

Identification of protein glycosylation sites is analytically challenging due to the diverse glycan structures associated with a glycoprotein. Mass spectrometry (MS)-based identification and characterization of glycoproteins has been achieved predominantly with the bottom-up approach, which typically involves the enzymatic cleavage of proteins to peptides prior to LC/MS or LC/MS/MS analysis. However, the process can be challenging due to the structural variations and steric hindrance imposed by the attached glycans. Alternatives to conventional heating protocols, that increase the rate of enzymatic cleavage of glycoproteins, may aid in addressing these challenges. An enzymatic digestion of a glycoprotein can be accelerated and made more efficient through microwave-assisted digestion. In this paper, a systematic study was conducted to explore the efficiency of microwave-assisted enzymatic (trypsin) digestion (MAED) of glycoproteins as compared with the conventional method. In addition, the optimum experimental parameters for the digestion such as temperature, reaction time, and microwave radiation power were investigated. It was determined that efficient tryptic digestion of glycoproteins was attained in 15 min, allowing comparable if not better sequence coverage through LC/MS/MS analysis. Optimum tryptic cleavage was achieved at 45°C irrespective of the size and complexity of the glycoprotein. Moreover, MAED allowed the detection and identification of more peptides and subsequently higher sequence coverage for all model glycoprotein. MAED also did not appear to prompt a loss or partial cleavage of the glycan moieties attached to the peptide backbones.

Pressure-assisted tryptic digestion using a syringe.

A simple and effective digestion method was developed using a syringe. A 3 mL syringe was used to apply a pressure of 6 atm to expedite tryptic digestion. Application of a pressure of 6 atm during digestion resulted in better digestion efficiency than digestion under atmospheric pressure. The protein peaks in the matrix-assisted laser desorption/ionization mass spectra of three model proteins (cytochrome c, horse heart myoglobin, and bovine serum albumin (BSA)) completely disappeared within 30 min at 37 degrees C under a pressure of 6 atm, with greater numbers of peptides observed in 30 min pressure-assisted digestion than in overnight atmospheric pressure digestion. This is mostly due to the miscleaved peptides. Similar sequence coverages were obtained for 30 min pressure-assisted digestion and overnight atmospheric pressure digestion of the three model proteins (92% vs. 88% for cytochrome c, 100% vs. 97% for horse heart myoglobin, and 53% vs. 53% for BSA).

Mass spectrometry characterization of species-specific peptides from arginine kinase for the identification of commercially relevant shrimp species.

The identification of commercial shrimp species is a relevant issue to ensure correct labeling, maintain consumer confidence and enhance the knowledge of the captured species, benefiting both, fisheries and manufacturers. A proteomic approach, based on 2DE, tryptic in-gel digestion, MALDI-TOF MS, and ESI-MS/MS analyses, is proposed for the identification of shrimp species with commercial interest. MALDI-TOF peptide mass fingerprint from arginine kinase tryptic digests were used for the identification of seven commercial, closely related species of Decapoda shrimps. Further identification and characterization of these peptides was performed by CID on an ESI-IT instrument, database search and de novo sequence interpretation, paying special attention to differential, species-specific peptides. Fisheries and manufacturers may take advantage of this methodology as a tool for a rapid and effective seafood product identification and authentication, providing and guaranteeing the quality and safety of the foodstuffs to consumers.

High-performance hardware implementation of a parallel database search engine for real-time peptide mass fingerprinting.

MOTIVATION: Peptide mass fingerprinting (PMF) is a method for protein identification in which a protein is fragmented by a defined cleavage protocol (usually proteolysis with trypsin), and the masses of these products constitute a 'fingerprint' that can be searched against theoretical fingerprints of all known proteins. In the first stage of PMF, the raw mass spectrometric data are processed to generate a peptide mass list. In the second stage this protein fingerprint is used to search a database of known proteins for the best protein match. Although current software solutions can typically deliver a match in a relatively short time, a system that can find a match in real time could change the way in which PMF is deployed and presented. In a paper published earlier we presented a hardware design of a raw mass spectra processor that, when implemented in Field Programmable Gate Array (FPGA) hardware, achieves almost 170-fold speed gain relative to a conventional software implementation running on a dual processor server. In this article we present a complementary hardware realization of a parallel database search engine that, when running on a Xilinx Virtex 2 FPGA at 100 MHz, delivers 1800-fold speed-up compared with an equivalent C software routine, running on a 3.06 GHz Xeon workstation. The inherent scalability of the design means that processing speed can be multiplied by deploying the design on multiple FPGAs. The database search processor and the mass spectra processor, running on a reconfigurable computing platform, provide a complete real-time PMF protein identification solution.

Centrifugal methods and devices for rapid in-gel digestion of proteins.

Modern proteomic research frequently relies upon separation of proteins in a polyacrylamide gel matrix followed by in-gel enzymatic digestion and extraction of peptides for subsequent analysis by MS. In this work, we propose a novel semi-automated method of mechanical processing of gel bands by passing these bands through a specially designed centrifugal device termed a Gel Shredder prior to digestion and extraction of peptides. Such a device allows integrated washing, destaining and shredding of gel bands into uniform blocks of controlled size, approximately 150-300 microm, prior to the enzymatic digestion and extraction of peptides. Shredding into uniform blocks increases the surface area of the gel pieces and promotes improved gel rehydration, allowing improved diffusion of the proteolytic enzymes and solvent into the gel lattice. We demonstrate that the new method substantially reduces the time spent on tedious manual handling of gel bands, while minimizing the risk of sample contamination. The performance of the Gel Shredder has been compared with a conventional in-gel digestion protocol using several standard proteins and a complex proteomic sample in terms of relative quantitation by either MALDI-TOF/TOF or nanoLC-ESI IT-Fourier transformation ion cyclotron resonance MS. It is shown that significant time savings and improved peptide recovery can be obtained for many proteins using the Gel Shredder compared with the traditional in-gel digestion protocol.

Development of protein-detecting microarrays and related devices.

There is great interest in the development of devices capable of monitoring the levels and post-translational modification states of hundreds or thousands of proteins simultaneously. One way to do this would be to create protein-detecting microarrays roughly akin to the DNA microarrays that are used for genome-wide expression studies. Two major challenges must be addressed before practical devices of this type become available. One is the development of high-throughput methods for the isolation of protein-binding compounds that will act as capture molecules in the array. The second is the optimization of methods that register binding of target proteins to the immobilized ligands in a sensitive and quantitative fashion. Progress in these areas, and some of the challenges remaining, are reviewed in this article.

Protein and RNA dynamical fingerprinting.

Protein structural vibrations impact biology by steering the structure to functional intermediate states; enhancing tunneling events; and optimizing energy transfer. Strong water absorption and a broad continuous vibrational density of states have prevented optical identification of these vibrations. Recently spectroscopic signatures that change with functional state were measured using anisotropic terahertz microscopy. The technique however has complex sample positioning requirements and long measurement times, limiting access for the biomolecular community. Here we demonstrate that a simplified system increases spectroscopic structure to dynamically fingerprint biomacromolecules with a factor of 6 reduction in data acquisition time. Using this technique, polarization varying anisotropy terahertz microscopy, we show sensitivity to inhibitor binding and unique vibrational spectra for several proteins and an RNA G-quadruplex. The technique's sensitivity to anisotropic absorbance and birefringence provides rapid assessment of macromolecular dynamics that impact biology.