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Liquid chromatography-mass spectrometry (LC-MS) intact mass analysis and LC-MS/MS peptide mapping are decisional assays for developing biological drugs and other commercial protein products. Certain PTM types, such as truncation and oxidation, increase the difficulty of precise proteoform characterization owing to inherent limitations in peptide and intact protein analyses. Top-down MS (TDMS) can resolve this ambiguity via fragmentation of specific proteoforms. We leveraged the strengths of flow-programmed (fp) denaturing online buffer exchange (dOBE) chromatography, including robust automation, relatively high ESI sensitivity, and long MS/MS window time, to support a TDMS platform for industrial protein characterization. We tested data-dependent (DDA) and targeted strategies using 14 different MS/MS scan types featuring combinations of collisional- and electron-based fragmentation as well as proton transfer charge reduction. This large, focused dataset was processed using a new software platform, named TDAcquireX, that improves proteoform characterization through TDMS data aggregation. A DDA-based workflow provided objective identification of αLac truncation proteoforms with a two-termini clipping search. A targeted TDMS workflow facilitated the characterization of αLac oxidation positional isomers. This strategy relied on using sliding window-based fragment ion deconvolution to generate composite proteoform spectral match (cPrSM) results amenable to fragment noise filtering, which is a fundamental enhancement relevant to TDMS applications generally., (© 2024 Wiley‐VCH GmbH.)

Peptide mapping by liquid chromatography-mass spectrometry is the gold standard to characterize post-translational modifications (PTMs) and disulfide bonds. The structural integrity, heterogeneity, and quality of biotherapeutic proteins are evaluated via reduced and non-reduced peptide mapping methods. However, non-enzymatic artifacts are often induced during sample preparation when alkaline pH conditions are used. To minimize these artifacts, methods using various acidic pH conditions have been developed by multiple researchers. However, these may lead to missed and non-specific cleavages during the analysis. In this study, an improved reduced and non-reduced peptide mapping method has been proposed to characterize endogenous chemical modifications and native disulfide bonds of monoclonal antibody-based products. Solubilization has been carried out at acidic pH conditions under high temperature, followed by the addition of tris (2-carboxyethyl) phosphine as a reducing agent and a low alkylating agent. It was observed that the non-enzymatic PTMs and non-native disulfide scrambled peptides significantly reduced under trypsin plus endoproteinase Lys-C digestion conditions at acidic pH as compared to the traditional methods. The results demonstrate that the proposed reduced and non-reduced peptide mapping method using trypsin plus Lys-C could identify and quantify various chemical and disulfide heterogeneities with minimal artifacts., (© 2022 Wiley-VCH GmbH.)

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., (© 2020 Wiley-VCH GmbH.)

A unique peptide based search algorithm for identification of protein mixture using PMF is proposed. The proposed search algorithm utilizes binary search and heapsort programs to generate frequency chart depicting the unique peptides corresponding to all proteins in a proteome. The use of binary search program significantly reduces the time for frequency chart preparation to ∼2 s for a proteome comprising ∼23 000 proteins. The algorithm was applied to a three-protein mixture identification, host cell protein (HCP) analysis, and a simulation-generated data set. It was found that the algorithm could identify at least one unique peptide of a protein even in the presence of fourfold higher concentration of another protein. In addition, two HCPs that are known to be difficult to remove were missed by MS/MS approach and were exclusively identified using the presented algorithm. Thus, the proposed algorithm when used along with standard proteomic approaches present avenues for enhanced protein identification efficiency, particularly for applications such as HCP analysis in biopharmaceutical research, where identification of low-abundance proteins are generally not achieved due to dynamic range limitations between the target product and HCPs., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Fragmentation in protein-based molecules continues to be a challenge during manufacturing and storage, and requires an appropriate control strategy to ensure purity and integrity of the drug product. Electrophoretic and chromatographic methods are commonly used for monitoring the fragments. However, size-exclusion chromatography often suffers from low resolution of low molecular weight fragments. Electrophoretic methods like CE-SDS are not compatible with enriching fragments for additional characterization tests such as MS. These limitations may result in inadequate control strategy for monitoring and characterizing fragments for protein-based molecules. Capillary western blotting was used in this study as an orthogonal method for characterization of fragments in an IgG1 antibody under reduced conditions. To achieve a comprehensive mapping of various fragments generated by thermal stress, capillary western profiles were generated using recognition antibodies for IgG kappa (κ) light chain, Fc, and Fab regions that enabled unambiguous fragment identification. Additionally, three different enzymatic digestion methods (IdeS, PNGase F, and IgdE) were applied coupled with capillary western blotting for clip identifications. Finally, complementary data collected using traditional chromatographic and electrophoretic methods allowed to establish a comparison of analytical profiles with an added benefit of fragment identification offered by capillary western profiling. In addition to various Fc and Fab-related low molecular weight fragments, a non-reducible thio-ether linked 75 kDa HL fragment was also identified., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In the present study, we have designed a laser-induced fluorescence (LIF) based instrumentation and developed a sensitive methodology for the effective separation, visualization, identification and analysis of proteins on a single platform. In this method, intrinsic fluorescence spectra of proteins were detected after separation on 1 or 2 dimensional Sodium Dodecyl Sulfate-Tris(2-carboxyethyl)phosphine (SDS-TCEP) polyacrylamide gel electrophoresis (PAGE) and the data were analyzed. The MATLAB assisted software was designed for the development of PAGE fingerprint for the visualization of protein after 1- and 2-dimensional protein separation. These provided objective parameters of intrinsic fluorescence intensity, emission peak, molecular weight and isoelectric point using a single platform. Further, the current architecture could differentiate the overlapping proteins in the PAGE gels which otherwise were not identifiable by conventional staining, imaging and tagging methods. Categorization of the proteins based on the presence or absence of tyrosine or tryptophan residues and assigning the corresponding emission peaks (309-356 nm) with pseudo colors allowed the detection of proportion of proteins within the given spectrum. The present methodology doesn't use stains or tags, hence amenable to couple with mass spectroscopic measurements. This technique may have relevance in the field of proteomics that is used for innumerable applications., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Strategically fabricated theranostic nanocarrier delivery system is an unmet need in personalized medicine. Herein, this study reports a versatile folate receptor (FR) targeted nanoenvelope delivery system (TNEDS) fabricated with gold core silica shell followed by chitosan-folic acid conjugate surface functionalization by for precise loading of doxorubicin (Dox), resembled as Au@SiO 2 -Dox-CS-FA. TNEDS possesses up to 90% Dox loading efficiency and internalized through endocytosis pathway leading to pH and redox-sensitive release kinetics. The superior FR-targeted cytotoxicity is evaluated by the nanocarrier in comparison with US Food and Drug Administration (FDA)-approved liposomal Dox conjugate, Lipodox. Moreover, TNEDS exhibits theranostic features through caspase-mediated apoptosis and envisages high surface plasmon resonance enabling the nanoconstruct as a promising surface enhanced Raman scattering (SERS) nanotag. Minuscule changes in the biochemical components inside cells exerted by the TNEDS along with the Dox release are evaluated explicitly in a time-dependent fashion using bimodal SERS/fluorescence nanoprobe. Finally, TNEDS displays superior antitumor response in FR-positive ascites as well as solid tumor syngraft mouse models. Therefore, this futuristic TNEDS is expected to be a potential alternative as a clinically relevant theranostic nanomedicine to effectively combat neoplasia., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Discriminating different rhinitis cases can sometimes be difficult as the diagnostic criteria used to identify the various subgroups are not always unambiguous. The nasal fluid (NF) highly reflects the pathophysiology of these inflammatory diseases. However, its collection, as nasal lavage fluid, may cause discomfort. Due to the non-invasiveness and rapidity of collection, nasal swab might represent an alternative to overcome these problems and also an ideal source of biomarkers. In this study, we demonstrate that the combined use of mesoporous silica (MPS) with MALDI-TOF MS allows the rapid detection of differential nasal peptide profiles from nasal swabs of healthy (H), allergic rhinitis (AR) and non-allergic rhinitis (NAR) subjects. NF peptides from nasal swabs were captured by the mean of MPS then profiled by MALDI-TOF MS. As a proof-of-principle, we also explored the ability of our platform to discriminate between nasal swabs of patients with AR and NAR, and between these groups and H controls. Four peaks resulted differentially expressed between NAR and AR, two peaks discriminated AR from H while one peak segregated NAR from H group. Therefore, peptides selected and enriched by our platform could form a part of a diagnostic ''rhinomic'' profile of the allergic and non-allergic patients., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Nanopore technology has become a highly sensitive and powerful tool for single molecule sensing of chemicals and biopolymers. Protein pores have the advantages of size amenability, channel homogeneity, and fabrication reproducibility. But most well-studied protein pores for sensing are too small for passage of peptide analytes that are typically a few nanometers in dimension. The funnel-shaped channel of bacteriophage phi29 DNA packaging motor has previously been inserted into a lipid membrane to serve as a larger pore with a narrowest N-terminal constriction of 3.6 nm and a wider C-terminal end of 6 nm. Here, the utility of phi29 motor channel for fingerprinting of various peptides using single molecule electrophysiological assays is reported. The translocation of peptides is proved unequivocally by single molecule fluorescence imaging. Current blockage percentage and distinctive current signatures are used to distinguish peptides with high confidence. Each peptide generated one or two distinct current blockage peaks, serving as typical fingerprint for each peptide. The oligomeric states of peptides can also be studied in real time at single molecule level. The results demonstrate the potential for further development of phi29 motor channel for detection of disease-associated peptide biomarkers., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Current approaches to protein identification rely heavily on database matching of fragmentation spectra or precursor peptide ions. We have developed a method for MALDI TOF-TOF instrumentation that uses peptide masses and their measurement errors to confirm protein identifications from a first pass MS/MS database search. The method uses MS1-level spectral data that have heretofore been ignored by most search engines. This approach uses the distribution of mass errors of peptide matches in the MS1 spectrum to develop a probability model that is independent of the MS/MS database search identifications. Peptide mass matches can come from both precursor ions that have been fragmented as well as those that are tentatively identified by accurate mass alone. This additional corroboration enables us to confirm protein identifications to MS/MS-based scores that are otherwise considered to be only of moderate quality. Straightforward and easily applicable to current proteomic analyses, this tool termed "ProteinProcessor" provides a robust and invaluable addition to current protein identification tools., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In pathogenic bacteria, posttranslationally modified proteins have been found to promote bacterial survival, replication, and evasion from the host immune system. In the human pathogen Neisseria meningitidis, the protein PilE (15-18 kDa) is the major building block of type IV pili, extracellular filamentous organelles that play a major role in mediating pathogenesis. Previous reports have shown that PilE can be expressed as a number of different proteoforms, each harboring its own set of PTMs and that specific proteoforms are key in promoting bacterial virulence. Efficient tools that allow complete PTM mapping of proteins involved in bacterial infection are therefore strongly needed. As we show in this study, a simple combination of mass profiling and bottom-up proteomics is fundamentally unable to achieve this goal when more than two proteoforms are present simultaneously. In a N. meningitidis strain isolated from a patient with meningitis, mass profiling revealed the presence of four major proteoforms of PilE, in a 1:1:1:1 ratio. Due to the complexity of the sample, a top-down approach was required to achieve complete PTM mapping for all four proteoforms, highlighting an unprecedented extent of glycosylation. Top-down MS therefore appears to be a promising tool for the analysis of highly posttranslationally modified proteins involved in bacterial virulence., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Gan et al. (Proteomics 2013, 13, 3117-3123) described a new "macropore" protocol for effective protein digestion by trypsin suitable for a wide range of pH including acidic pH. It was effective not only in experiments with solutions of a model protein (myoglobin), but also with a subfraction of rat liver cytosol. This significantly simplifies and accelerates protein digestion procedures for subsequent MS. However, further studies are needed to find limits of experimental applicability of the described protocol in proteomics., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Amino-functionalized macroporous silica foam (NH2 -MOSF) has been developed as a host reactor to realize highly efficient proteolysis in acidic solutions where normal tryptic reactions cannot occur. The digestion protocol consists simply of adding the functionalized NH2 -MOSF into the protein and trypsin solutions without altering the bulk pH or preloading the enzymes on the materials. With this protocol, digestion of sample fractions from LC can be efficiently realized in the acidic solutions directly. Digestion of a protein fraction extracted from rat liver tissue after LC separation was performed to illustrate this principle, where 103 proteins were successfully identified at pH 3 after 1.5 h of tryptic digestion., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

To obtain a comprehensive understanding of proteins involved in excitation-contraction coupling, a catalog of proteins from sarcoplasmic reticulum (SR) membrane fractions of New Zealand white rabbit skeletal muscle was analyzed by an optimized shotgun proteomic method. Light and heavy SR membrane fractions were obtained by nonlinear sucrose gradient centrifugation and separated by 1DE followed by a highly reproducible, automated LC-MS/MS on the hybrid linear ion trap (LTQ) Orbitrap mass spectrometer. By integrating as low as 1% false discovery rate as one of the features for quality control method, 483 proteins were identified from both of the two independent SR preparations. Proteins involved in calcium release unit complex, including ryanodine receptor 1, dihydropyridine receptor, calmodulin, triadin, junctin, and calsequestrin, were all detected, which offered validation for this protein identification method. Rigorous bioinformatics analysis was performed. Protein pI value, molecular weight range, hydrophobicity index, and transmembrane region were calculated using bioinformatics softwares. Eighty-three proteins were classified as hydrophobic proteins and 175 proteins were recognized as membrane proteins. Based on the proteomic analysis results, we found as the first time that not only transverse tubule but also mitochondrion physically connected to SR. The complete mapping of these proteomes may help in the elucidation of the process of excitation-contraction coupling and excitation-metabolism coupling., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The rabbit is an important species as both a production animal and as a model organism in physiology, pharmaceutical, and numerous other studies. Similar to other species, the rabbit skeletal muscle proteome has been characterized, first using 2DE mapping and more recently using high-throughput shotgun proteomics. This article is a commentary on "Shotgun proteomics analysis of the sarcoplasmic reticulum preparations from rabbit skeletal muscle" (Z. Liu et al., Proteomics, 2013, 13, 2335-2338). Herein, we present the reasons why the manuscript is of high relevance to three major fields: farm animal, rabbit, and muscle/meat proteomics., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Immobilized proteolytic enzymes present several advantages over their soluble form, not the least of which is suppression of autoproteolysis peaks even at high enzyme-to-substrate ratios. We have made immobilized chymotrypsin by directly crosslinking it with glutaraldehyde to produce polymeric particles. Digestion of two model substrates using the particles was followed by CE peptide mapping with detection by UV absorbance or LIF. Results showed that autoproteolysis was highly suppressed and that different storage conditions of the particles in the short term (24 h) did not affect digestion of denatured BSA. As well, the chymotrypsin particles were indifferent to the presence of fluorescein groups on a casein substrate. Glutaraldehyde crosslinking of chymotrypsin inside a fused silica capillary column to make an immobilized enzyme reactor (IMER) was achieved in a series of reagent addition and washing steps, entirely automated using a commercial CE instrument. Digestion of myoglobin in the IMER for 30 min at 37°C followed by peptide mapping by CE-MS of the collected digest allowed identification of 17 chymotryptic peptides of myoglobin, or 83% primary sequence coverage., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In this work we report for the first time a post-translational modification of PII homologues from the Archaea Domain. Haloferax mediterranei is the first haloarchaea whose PII proteins have been studied, it possesses two of them (GlnK1 and GlnK2 ), both encoded adjacent to a gene for the ammonia transporter Amt. An approach based on 2DE, anti-GlnK immunoblot and peptide mass fingerprint (MALDI-TOF-MS) of the reactive spots showed that GlnK proteins in H. mediterranei are post-translationally uridylylated. A third spot with lower pI suggests the existence of a non-descript post-translational modification in this protein family., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Ultrahigh resolution MALDI-FT-ICR profiles were obtained from human serum samples that were processed using a fully automated RPC18-based magnetic bead method. Proteins were profiled from m/z value 6630 with a resolving power of 73 000 up to m/z value 12 600 with a resolving power of 37 000. In this study, a detailed evaluation was performed of the isoforms of apolipoprotein C-III, i.e. the different mucin-type core 1 O-glycans with the addition of one or two sialic acid residues. The MALDI-FT-ICR profiles are discussed with regard to reproducibility of the signal intensities as well as the accurate mass measurements. ESI-FT-ICR-MS/MS analyses of the same serum samples were performed to confirm the identity of apolipoprotein C-III glycoforms., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The analysis of peptides and proteins in complex biological systems has greatly improved over the last decade. State-of-the-art mass spectrometric instruments combined with adequate software tools allow for more and more comprehensive proteome analyses. Most proteome-wide studies focus on the analysis of unmodified proteins or look at selected modifications only. However, spectral information of protein modifications, chemically induced through sample preparation or post-translationally attached in biological pathways is acquired as a significant, yet disregarded, fraction of tandem spectra in most discovery studies. We present a new computational pipeline, PTMeta, to uncover information of modifications attached to peptides. We use modification prescanning to pinpoint the most abundant potential modifications, followed by extensive database search and a statistical framework to combine results from database search runs with different modification settings., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

LC-ESI/MS/MS-based shotgun proteomics is currently the most commonly used approach for the identification and quantification of proteins in large-scale studies of biomarker discovery. In the past several years, the shotgun proteomics technologies have been refined toward further enhancement of proteome coverage. In the complex series of protocols involved in shotgun proteomics, however, loss of proteolytic peptides during the lyophilization step prior to the LC/MS/MS injection has been relatively neglected despite the fact that the dissolution of the hydrophobic peptides in lyophilized samples is difficult in 0.05-0.1% TFA or formic acid, causing substantial loss of precious peptide samples. In order to prevent the loss of peptide samples during this step, we devised a new protocol using Invitrosol (IVS), a commercially available surfactant compatible with ESI-MS; by dissolving the lyophilized peptides in IVS, we show improved recovery of hydrophobic peptides, leading to enhanced coverage of proteome. Thus, the use of IVS in the recovery step of lyophilized peptides will help the shotgun proteomics analysis by expanding the proteome coverage, which would significantly promote the discovery and development of new diagnostic markers and therapeutic targets., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We report the first survey of the dromedary camel urinary proteome. Proteins retained from ultrafiltration of urine were analysed by GeLC-MS/MS (SDS-PAGE followed by LC-MS/MS). In the absence of a complete camel genome sequence, the number of protein identifications was maximised by searching three primary sequence databases: Swiss-Prot, alpaca and camel EST. This search strategy enabled the identification of 1274 peptide sequences, of which 735 were found in at least two independent samples. Functional annotations for proteins identified from alpaca and camel EST sequences were mapped from basic local alignment search tool (protein) searches. These 735 peptides, which included many novel sequences found only in the camel EST database, were grouped to 147 protein descriptors. Gene ontology term analysis of human proteins with sequence similarity showed that camel urine may be particularly enriched in proteins from extracellular compartments and vesicles, and with functions that include carbohydrate-binding and peptidase inhibitor activities. If their biological functions are conserved between species, many of the camel urinary proteins could be involved in various stress and immune responses, and some may have antimicrobial activities., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Traditional methods for bacterial identification include Gram staining, culturing, and biochemical assays for phenotypic characterization of the causative organism. These methods can be time-consuming because they require in vitro cultivation of the microorganisms. Recently, however, it has become possible to obtain chemical profiles for lipids, peptides, and proteins that are present in an intact organism, particularly now that new developments have been made for the efficient ionization of biomolecules. MS has therefore become the state-of-the-art technology for microorganism identification in microbiological clinical diagnosis. Here, we introduce an innovative sample preparation method for nonculture-based identification of bacteria in milk. The technique detects characteristic profiles of intact proteins (mostly ribosomal) with the recently introduced MALDI Sepsityper(TM) Kit followed by MALDI-MS. In combination with a dedicated bioinformatics software tool for databank matching, the method allows for almost real-time and reliable genus and species identification. We demonstrate the sensitivity of this protocol by experimentally contaminating pasteurized and homogenized whole milk samples with bacterial loads of 10(3) -10(8) colony-forming units (cfu) of laboratory strains of Escherichia coli, Enterococcus faecalis, and Staphylococcus aureus. For milk samples contaminated with a lower bacterial load (10(4) cfu mL(-1) ), bacterial identification could be performed after initial incubation at 37°C for 4 h. The sensitivity of the method may be influenced by the bacterial species and count, and therefore, it must be optimized for the specific application. The proposed use of protein markers for nonculture-based bacterial identification allows for high-throughput detection of pathogens present in milk samples. This method could therefore be useful in the veterinary practice and in the dairy industry, such as for the diagnosis of subclinical mastitis and for the sanitary monitoring of raw and processed milk products., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We report a technique for isolation and solubilization of intermediate filament (IF) proteins from colonic biopsies compatible with both gel electrophoresis and liquid chromatography "shotgun" proteomics using mass spectrometry (MS). This is important because changes in the IF proteome, particularly in keratin expression and modification, are noted in colonic mucosa of patients with colorectal cancer. Though keratins have traditionally been dissolved in high concentration of urea, the latter solvent precludes efficient proteolytic digestion by trypsin prior to gel-free LC-MS/MS approaches. The extraction of cytoskeletal proteins was initially evaluated using MCF-7 cancer cell lines using a published, differential detergent solubilization protocol. IF proteins were extracted from colonic biopsies using a combination of homogenization and sonication. Since comparable efficiency of solubilization was noted on the extracted IF from cell lines between urea and guanidine hydrochloride (GuHCl) in triethylammonium bicarbonate buffer, isolated proteins from endoscopic biopsies were solubilized in GuHCl. Using immunoblotting techniques, we successfully demonstrated isolation of keratins and preservation of posttranslational modifications (phosphorylation, acetylation). Dissolved proteins were tryptically digested and peptides analyzed by MS, showing the functionality of the workflow in shotgun proteomic applications, specifically compatibility of the workflow for isobaric tagging relative and absolute quantification based quantitation approaches., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Tryptic peptide mapping is routinely used in the biotech industry to confirm primary sequence, cell line stability, and to analyze posttranslational modifications. Peptide analysis is generally done by reverse phase liquid chromatography with UV or mass spectrometric detection. This method provides excellent resolution and sequence coverage. However, traditional methods are slow, and generally cannot detect small, hydrophilic peptides due to coelution with the column void volume. In this work, complementary CE-MS peptide analysis methods have been developed. The analyses are performed on a traditional CE-MS instrument with a sheath interface, and also on a novel sheathless interface that promises improved resolution and limit of detection. The methods were performed on a tryptic digest of a therapeutic monoclonal antibody for which LC-MS detects 97% sequence coverage. The 3% not covered consists of 11 peptides containing three amino acids or fewer, including two in the critical complementarity binding domain. Without further processing, the same tryptic digest was analyzed by CE-MS. Separation and detection of the 11 small peptides was achieved on CE-MS systems with both interfaces. The sheathless system produced better peak capacity and gave mass spectra with significantly less noise, while the sheath system proved to have better repeatability., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Two different neutral nonpolar monolithic columns series (designated as A and B columns series) each consisting of three columns at varying n-alkyl chain length were prepared by the copolymerization of the functional monomers C8-methacrylate, C12-acrylate, or C16-methacrylate with the cross-linking monomer pentaerythritol triacrylate (PETA) to yield monoliths with surface bound C8, C12, and C16 chains. In the A columns series, the composition of the functional monomers and crosslinker was adjusted to yield comparable chromatographic retention regardless of the alkyl chain length. In the B columns series, the composition of the functional monomers and crosslinker was kept constant yielding chromatographic retention, which increased as expected in the order of increasing the n-alkyl chain length. Due to their direct influences on the monolith porosity and retention energetic, the nature and composition of the monomers at a given porogen composition have largely affected the solute's mass transfer characteristics and sorption kinetics, as assessed by the van Deemter plots and separation efficiencies. The C16-monolith of the A series yielded the highest separation efficiency toward small solutes, but the A columns series were inadequate for protein separation. The C8-monolith of the B series provided the best separation efficiency for proteins while for tryptic peptide mapping, the C16-monolith of the A series seems to provide the best separation. For large protein molecules, the energetically "softer" C8 surface allowed faster sorption kinetics and in turn improved efficiency, while an energetically "harder" C16 surface favored better separation of the smaller size peptide solutes., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Recently, we found that hemoglobin (Hb) could be re-released from live erythrocytes during electrophoresis release test (ERT). The re-released Hb displays single-band and multiple-band re-release types, but its exact mechanism is not well understood. In this article, the protein components of the single-band re-released Hb were examined. First, the re-released band of erythrocytes and the corresponding band of hemolysate, which was used as control, were cut out from starch-agarose mixed gel. Next, proteins were recovered from the starch-agarose mixed gel by freeze-thaw method. After condensing in a vacuum freeze drier, the samples were loaded onto a 5-12% SDS-PAGE. After electrophoresis, three protein bands (16, 28.9, and 29.3 kDa) emerged from the erythrocytes re-released Hb single-band (R-R), but only one band (29.3 kDa) emerged from the corresponding hemolysate control band (H-R). Finally, these bands were analyzed by MALDI-TOF MS. The results showed that these proteins were beta-globin (16 kDa), carbonic anhydrase 1 (CA1, 28.9 kDa), and carbonic anhydrase 2 (CA2, 29.3 kDa). Because CA2 exists in both erythrocytes re-released band and hemolysate control band, we conclude that the single-band re-released Hb is mainly composed of HbA and CA1. Studying the possible interaction between HbA and CA1 will help us further understand the in vivo function of Hb., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

RUNX1 is essential for definitive hematopoiesis and T-cell differentiation. It has been shown that RUNX1 is phosphorylated at specific serine and threonine residues by several kinase families. However, it remains unclear whether RUNX1 phosphorylation is absolutely required for its biological functions. Here, we evaluated hematopoietic activities of RUNX1 mutants with serine (S)/threonine (T) to alanine (A), aspartic acid (D), or glutamic acid (E) mutations at phosphorylation sites using primary culture systems. Consistent with the results of knockin mice, RUNX1-2A, carrying two phospho-deficient mutations at S276 and S293, retained hematopoietic activity. RUNX1-4A, carrying four mutations at S276, S293, T300, and S303, showed impaired T-cell differentiation activity, but retained the ability to rescue the defective early hematopoiesis of Runx1-deficient cells. Notably, RUNX1-5A, carrying five mutations at S276, S293, T300, S303, and S462, completely lost its hematopoietic activity. In contrast, the phospho-mimic proteins RUNX1-4D/E and RUNX1-5D/E exhibited normal function. Our study identifies multiple phosphorylation sites that are indispensable for RUNX1 activity in hematopoiesis., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In this review, we report the evolution on experimental conditions for the analysis of normal urine based on combinatorial peptide ligand library (CPLL) treatment and successive 2-DE and 2-DE/MS analysis. The main topics are (i) definition of the urine sample requirements, (ii) optimization of the urine/ligand ratio, (iii) essay conditions, (iv) en bloc elution. Overall, normal urine protein composition as studied by 2-DE includes over 2600 spots. Relevant data on inter and intraessay reproducibility obtained by the analysis of different normal urines repeated several times are also here presented. We found a 73% reproducibility upon analysis of the same sample and 68% correspondence of protein composition among different normal urine samples. Based on the above results, we are completing the characterization with LC-MS of 249 spots. The composition of normal urine proteins after CPLLs is finally shown with the indication of those spots which are currently under identification. This map will be completed in a near future; in the meantime this would represent the basic reference sample for newly developed studies on human diseases., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Venom from the scorpion Pandinus cavimanus was obtained by electrical stimulation of the telson (stinger). Total venom was toxic to crickets at 7-30 μg and a paralysis or lethal effect was observed at 30 μg of venom (death at 1.5 μg/mg of cricket). Electrophysiological analyses showed cytolytic activity of total venom on oocytes at 7 μg. HPLC allowed separation of the venom components. A total of 38 fractions from total venom were tested on voltage-gated Na(+) and K(+) channels. Some fractions block K(+) currents in different degrees. By using MS analysis, we obtained more than 700 different molecular masses from telson and venom fractions (by LC-MS/MS and MALDI-TOF MS analyses). The number of disulfide bridges of the telson components was determined. A cDNA library from P. cavimanus scorpion was constructed and a random sequencing screening of transcripts was conducted. Different clones were obtained and were analyzed by bioinformatics tools. Our results reveal information about new genes related to some cellular processes and genes involved in venom gland functions (toxins, phospholipases and antimicrobial peptides). Expressed sequence tags from venom glands provide complementary information to MS and reveal undescribed components related to the biological activity of the venom., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

SDS is mostly used to enhance the solubilization and extraction of membrane proteins due to its strong detergency and low cost. Nevertheless, SDS interferes with the subsequent procedures and needs to be removed from the samples. In this work, a special gradient gel electrophoresis (GGE) system was developed to remove SDS from the SDS-solubilized protein samples. As a proof-of-principle experiment, the GGE system was designed to be composed of an agarose loading layer, six polyacrylamide fractionation layers with different concentrations and a high-concentration polyacrylamide sealing layer. The advantages of the GGE system are that it not only can electrophoretically remove SDS efficiently so that the protein loss resulted from the repeated gel washing after electrophoresis was avoided, but also can reduce the complexity of the sample, prevent the precipitation of proteins after loading and avoid the loss of proteins with low molecular weight during the electrophoresis. Using GGE system, about 85% of SDS in the sample and gel was electrophoretically removed and the proteins were fractionated. Compared with the two representative gel-based sample cleanup methods reported in literature, GGE-based strategy significantly improved the identification efficiency of proteins in terms of the number and coverage of the identified proteins., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Gender differences in obesity stem from metabolic and hormonal differences between sexes and contribute to differences between women and men in health risks attributable to obesity. We hypothesized that liver may be an ideal target for the evaluation of gender differences in obesity development in response to a high-fat diet (HFD). Therefore, to test this hypothesis, we performed a global proteome analysis in the liver of lean and obese rats of both genders who were fed an HFD through 2-DE combined with MALDI-TOF-MS. When rats were exposed to HFD, male rats gained more body weight with increased values of plasma biochemical parameters than female rats. Image analysis and further statistical analysis of a 2-DE protein map allowed for the detection and identification of 34 proteins that were significantly modulated in a gender-dependent manner. We found 19 proteins showing identical gender-different regulation in both normal diet (ND) and HFD. Five proteins also showed clear gender differences in both ND and HFD; however, their regulation modes in HFD were opposite to those in ND. Of particular interest, 10 proteins showed gender differences only in either ND or HFD rats. Present proteomic insight into gender-dimorphic protein modulation in liver would aid in the improvement of gender awareness in the health-care system and in implementation of evidence-based gender-specific clinical recommendations., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Knowledge of gender differences is important because nutritional recommendations on the basis of data collected using predominantly male subjects may not be valid for women. In the present study, we performed proteomic analysis in plasma of rats fed a high-fat diet (HFD) using 2-DE combined with MALDI-TOF-MS for analysis of differential regulation patterns between male and female plasma proteins. Male rats gained more body weight with increased values of biochemical parameters than female rats. Image analysis and further statistical analysis allowed detection and identification of 31 proteins that were significantly modulated in a gender-dependent manner in response to HFD. Those differential expressed proteins were classified into three groups based on their regulation patterns in response to diet and gender. Consequently, we found 13 proteins showing gender-different regulation in both normal diet (ND) and HFD, where 9 proteins showed identical regulation patterns (Group I) and 4 proteins exhibited opposite regulation mode (Group II) between the genders. Eighteen proteins showed no gender-difference but HFD-responsive regulation (Group III). Of these, Apo A-IV, CRP precursor, Hp precursor, and FGG showed a clear gender difference in both ND and HFD, with the same regulation patterns. Present proteomic research into gender-dimorphic protein modulation in plasma would aid in improvement of gender awareness in the health care system and in implementation of evidence-based gender-specific clinical recommendations., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Although there are now multiple methods for the analysis of membrane proteomes, there is relatively little systematic characterization of proteomic workflows for membrane proteins. The Asia Oceania Human Proteome Organisation (AOHUPO) has therefore embarked on a Membrane Proteomics Initiative (MPI) using a large range of workflows. Here, we describe the characterization of the MPI mouse liver microsomal membrane Standard using SDS-PAGE prior to in-gel tryptic digestion and LC-ESI-MS/MS. The Na(2) CO(3) wash followed by SDS-PAGE prior to in-gel tryptic digestion and LC-MS/MS strategy was effective for the detection of membrane proteins with 47.1% of the identified proteins being transmembrane proteins. Gene Ontology term enrichment analysis showed that biological processes involving transport, lipid metabolism, cell communication, cell adhesion, and cellular component organization were significantly enriched. Comparison of the present data with the previously published reports on mouse liver proteomes confirmed that the MPI Standard provides an excellent resource for the analysis of membrane proteins in the AOHUPO MPI., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

This article reports the results of a study carried out to evaluate the offline hyphenation of capillary zone electrophoresis with matrix-assisted lased desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) for the analysis of low-abundant complex samples, represented by the tryptic phosphorylated peptides of phosphoproteins, such as α-casein, β-casein, and fetuin. The proposed method employs a latex-coated capillary and consists in the online preconcentration of the tryptic peptides by a pH-mediated stacking method, their separation by capillary zone electrophoresis, and subsequent deposition of the separated analytes onto a MALDI target for their MS analysis. The online preconcentration method allows loading a large sample volume (∼150 nL), which is introduced into the capillary after the hydrodynamic injection of a short plug of 1.0 M ammonium hydroxide solution and is sandwiched between two plugs of the acidic background electrolyte solution (BGE) filling the capillary. The sample spotting of the separated analytes onto the MALDI target is performed either during or postseparation using an automatic spotting device connected to the exit of the separation capillary. The proposed method allows the separation and identification of multiphosphorylated peptides from other peptides and enables their identification at femtomole level with improved efficiency compared with LC approaches hyphenated to MS., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Studying fear extinction is a major topic in neuroscience. No information on systematic studies on the linkage of contextual fear conditioning (cFC) with hippocampal protein levels is available and we were therefore interested in protein differences between animals with poor and good extinction. cFC was carried out in C57BL/6J mice, hippocampi were taken and proteins were run on two-dimensional gel electrophoresis with subsequent quantification of protein spots. In-gel digestion with trypsin and identification by ion trap MS/MS (high-capacity ion trap) was used for the identification of significantly different hippocampal proteins between mice with good and poor performance of extinction. Signaling protein ras-related protein rab-7A and septin 8 levels were significantly higher in hippocampus of poor extinguishers, whereas ubiquitin carboxyterminal hydrolase isozyme L1 showed higher levels in animals with good extinction performance. A series of additional proteins showed significantly different levels between groups but the abovementioned were confirmed by immunoblotting. The abovementioned proteins have never been reported to be linked to extinction, memory, or learning and herein evidence for the involvement of several proteins in extinction mechanism as well as probably representing pharmaceutical targets is provided. Moreover, it is intriguing to demonstrate the differences between good and poor extinction performance at the protein level., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

β-Ga(2)O(3) is a wide-band-gap semiconductor having strong oxidation ability under light irradiation. Herein, the steel target plates modified with β-Ga(2)O(3) nanoparticles have been developed to carry out in-source photo-catalytic oxidative reactions for online peptide tagging during laser desorption/ionization mass spectrometry (LDI-MS) analysis. Under UV laser irradiation, β-Ga(2)O(3) can catalyze the photo-oxidation of 2-methoxyhydroquinone added to a sample mixture to 2-methoxy benzoquinone that can further react with the thiol groups of cysteine residues by Michael addition reaction. The tagging process leads to appearance of pairs of peaks with an m/z shift of 138.1Th. This online labelling strategy is demonstrated to be sensitive and efficient with a detection-limit at femtomole level. Using the strategy, the information on cysteine content in peptides can be obtained together with peptide mass, therefore constraining the database searching for an advanced identification of cysteine-containing proteins from protein mixtures. The current peptide online tagging method can be important for specific analysis of cysteine-containing proteins especially the low-abundant ones that cannot be completely isolated from other high-abundant non-cysteine-proteins., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Proteases are critical in many physiological processes and the human genome encodes for 566 predicted proteolytic enzymes. Therefore, there is great interest in identifying and characterizing physiologic protease-substrate relationships. The coagulation cascade is a well-described network of serine proteases. However, new interactions of the coagulation cascade with other biological pathways have been discovered only recently. Therefore, we hypothesized that a non-biased protease degradomics analysis of the physiologic coagulation reaction would identify new interactions between the coagulation cascade and other pathways. We used the recently described PROTOMAP technique to profile the complete coagulation degradome. This analysis detected virtually all of the proteins of the coagulation cascade and identified a majority of the expected proteolytic events, suggesting significant coverage of the coagulation degradome. Multiple potential new proteolytic cleavages were detected, including two of transmembrane proteins that may be shed from the surface of blood cells. In addition, this analysis was able to identify several new potentially secreted proteins. A significant majority of the newly identified events were of proteins involved in innate immunity (complement and inflammation). This highlights potential new areas of crosstalk between these linked systems. Future studies will elucidate the details and functional consequences of these proteolytic events during coagulation., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The studies of neuropsychiatric disorders would be facilitated by enhanced knowledge of the dorsolateral prefrontal cortex (DLPFC) proteome. To construct a data set of human DLPFC proteins, protein extracts were prepared from 12 postmortem brains focussing on the DLPFC region (Brodmann area 9) and analyzed using a combined gel electrophoresis and shotgun mass spectrometry approach, featuring data-independent label-free nanoflow liquid chromatography mass spectrometry (nLC-MS(E)). The detected mass/time features were aligned and annotated using the results from ProteinLynx Global Server. The resulting data set comprised 488 unique and accurately identified proteins, with stringent identification by a minimum of two distinct peptides detected at least in >75% of samples. These proteins were involved predominantly in cytoskeletal architecture, metabolism, transcription/translation, and synaptic function. Combination of this data set with that obtained by our previous characterization of the same brain region results in a total of 755 unique proteins, making this the most comprehensive analysis of this important brain region to date., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Herein, we describe the development of a fully automatable technology that features online coupling of high-pH RP separation with conventional low-pH RP separation in a two-dimensional capillary liquid chromatography (2-D LC) system for shotgun proteomics analyses. The complete analysis comprises 13 separation cycles, each involving transfer of the eluate from the first-dimension, high-pH RP separation onto the second RP dimension for further separation. The solvent strength increases across the 13 fractions (cycles) to elute all peptides for further resolution on the second-dimension, low-pH RP separation, each under identical gradient-elution conditions. The total run time per analysis is 52 h. In triplicate analyses of a lysate of mouse embryonic fibroblasts, we used this technology to identify 2431 non-redundant proteins, of which 50% were observed in all three replicates. A comparison of RP-RP 2-D LC and strong cation exchange-RP 2-D LC analyses reveals that the two technologies identify primarily different peptides, thereby underscoring the differences in their separation chemistries., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

2-D DIGE was used to investigate 'fingerprint proteins' in biological medicines. A presumably non-originator human albumin was analysed, and the 2-D DIGE patterns of the non-genuine and the authentic product were compared. The products could be clearly distinguished based on the pattern of minor components, which represent plasma proteins and degradation products remaining in the final products after fractionation and purification. The approach demonstrated that 2-D DIGE is an excellent tool for the analysis of biologicals of different sources and for ensuring the identity and quality of blood products., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In this study, we evaluated a concatenated low pH (pH 3) and high pH (pH 10) reversed-phase liquid chromatography strategy as a first dimension for two-dimensional liquid chromatography tandem mass spectrometry ("shotgun") proteomic analysis of trypsin-digested human MCF10A cell sample. Compared with the more traditional strong cation exchange method, the use of concatenated high pH reversed-phase liquid chromatography as a first-dimension fractionation strategy resulted in 1.8- and 1.6-fold increases in the number of peptide and protein identifications (with two or more unique peptides), respectively. In addition to broader identifications, advantages of the concatenated high pH fractionation approach include improved protein sequence coverage, simplified sample processing, and reduced sample losses. The results demonstrate that the concatenated high pH reversed-phased strategy is an attractive alternative to strong cation exchange for two-dimensional shotgun proteomic analysis., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The need to find biomarkers for hepatobiliary diseases including cholangiocarcinoma (CCA) has led to an interest in using bile as a proximal fluid in biomarker discovery experiments, although there are inherent challenges both in its acquisition and analysis. The study described here greatly extends previous studies that have started to characterise the bile proteome. Bile from four patients with hilar CCA was depleted of albumin and immunoglobulin G and analysed by GeLC-MS/MS. The number of proteins identified per bile sample was between 378 and 741. Overall, the products of 813 unique genes were identified, considerably extending current knowledge of the malignant bile proteome. Of these, 268 were present in at least 3 out of 4 patients. This data set represents the largest catalogue of bile proteins to date and together with other studies in the literature constitutes an important prelude to the potential promise of expression proteomics and subsequent validation studies in CCA biomarker discovery., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We assessed whether the G-electrode-loading method (GELM) was helpful in the protein analysis. GELM in 2-DE was compared with the slip-loading, the in-gel rehydration and the cup-loading in 2-DE. GELM showed the best results for protein separation. A total of 14 spots that showed an increase with GELM were analyzed by MALDI-TOF MS. In GELM, all of these spots were identified with a high score and a high sequence coverage. A membrane-associated protein was identified and determined to have phosphorylated site. These tests show that GELM has several advantages for protein analysis compared with the traditional methods., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

To investigate the phosphorylation capability of serogroup A Neisseria meningitidis (MenA) and to implement our knowledge in meningococcal biology and in bacterial post-translational modifications, cell extracts were separated by 2-DE and 51 novel phosphoproteins were revealed by the use of the highly specific Ser/Thr/Tyr-phosphorylated proteins staining by Pro-Q Diamond and identified by MALDI-ToF/MS. Our results indicate that phosphorylation in MenA is comparable to that of other bacterial species. A first functional characterization of the identified modified proteins was also given, in order to understand their role in meningococcal physiopathology., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In this review we examine techniques, software, and statistical analyses used in label-free quantitative proteomics studies for area under the curve and spectral counting approaches. Recent advances in the field are discussed in an order that reflects a logical workflow design. Examples of studies that follow this design are presented to highlight the requirement for statistical assessment and further experiments to validate results from label-free quantitation. Limitations of label-free approaches are considered, label-free approaches are compared with labelling techniques, and forward-looking applications for label-free quantitative data are presented. We conclude that label-free quantitative proteomics is a reliable, versatile, and cost-effective alternative to labelled quantitation., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

Gastric juice is the most proximal fluid surrounding the stomach tissue. The analysis of gastric juice protein contents will thus be able to accurately reflect the pathophysiology of the stomach. This biological fluid is also a potential reservoir of secreted biomarkers in higher concentration as compared to the serum. Unlike the rest of the gastrointestinal fluids, there were very few studies reported on gastric juice proteome. To date, the proteins that routinely populate this biofluid are largely unknown. This is partly due to the technical difficulties in processing a sample that contains a collection of other gastrointestinal fluids, especially saliva. In this study, we attempt to profile the protein components of the gastric fluids from chronic gastritis patients using a direct shotgun proteomics approach. These data represent the first report of the proteome of human gastric juice with gastritis background.

A neutral naphthyl methacrylate-phenylene diacrylate-based monolith (NPM) was introduced for RP-CEC of various neutral and charged solute probes via hydrophobic and π interactions. The NPM column was prepared by the in situ polymerization of naphthyl methacrylate as the functional monomer and 1,4-phenylene diacrylate (PDA) as the crosslinker in a ternary porogenic solvent containing cyclohexanol, dodecanol and water. The NPM column exhibited cathodal EOF despite the fact that it was devoid of any fixed charges. NPM exhibited stronger EOF than its counterpart naphthyl methacrylate monolith (NMM) made from the in situ polymerization of naphthyl methacrylate and trimethylolpropane trimethacrylate (TRIM). As for NMM, it is believed that the EOF arises from the adsorption of mobile phase ions onto the monolith surface. The higher EOF exhibited by NPM may be attributed to the acrylate nature of PDA as compared to the methacrylate nature of TRIM, and therefore PDA has a higher binding capacity for mobile phase ions due to its higher polarity than TRIM. The adsorption of mobile phase ions together with the additional π interactions offered by the aromatic rings of the NPM matrix modulated solute retention and separation selectivity. The applications of NPM were demonstrated by the separation of a wide range of small and large solutes including peptides, tryptic peptide maps and proteins.

A neutral hydroxylated octadecyl monolith (ODM-OH) for reversed-phase capillary electrochromatography has been developed. The ODM-OH was prepared by the in situ polymerization of octadecyl acrylate and pentaerythritol triacrylate (PETA) in a ternary porogenic solvent. Pentaerythritol triacrylate possesses a hydroxyl functional group, which imparts the monolith with a hydrophilic group, thus the acronym ODM-OH. The ODM-OH column exhibited cathodal EOF over a wide range of pH and ACN concentration in the mobile phase despite the fact that it was devoid of any fixed charges. This ODM-OH monolith exhibited stronger EOF than its counterpart the ODM made from the in situ polymerization of octadecyl acrylate and trimethylolpropane trimethacrylate. Similar to ODM, it is believed that the EOF was due to the adsorption of ions from the mobile phase onto the surface of the monolith thus imparting the neutral monolithic column the zeta potential necessary to support the EOF. The higher EOF exhibited by ODM-OH was due to the presence of polar OH groups on its surface, which would favor stronger adsorption of ions from the mobile phase. The wide applications of the neutral ODM-OH column were demonstrated in the separation of a wide range of small and large solutes. As a typical result, the ODM-OH was able to separate proteins quite rapidly yielding 200,000 plates/m.