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Analytical requirements in the biopharmaceutical, pharmaceutical, and food industries, among several others, are more demanding than ever. Chromatographic techniques are great tools to acquire detailed information on a vast number of molecules and sample types. The present challenge in research and development (R&D), as well as in quality control (QC) laboratories, is to collect as much sample information as possible. However, even with the current one-dimensional (1D) analytical portfolio, it is not possible to fully ensure that all the relevant information from a sample has been captured. This article illustrates the power of an online two-dimensional liquid chromatographic (2D-LC) setup to unravel the complexity of biopharmaceutical and pharmaceutical samples. This technology tremendously increases the resolving power in all areas where LC is applied and drastically reduces the risk of missing information about the sample.

In this extended special feature to celebrate the 35th anniversary edition of LCGC Europe, leading figures from the separation science community explore contemporary trends in separation science and identify possible future developments.

Characterisation of highly glycosylated biopharmaceuticals by mass spectrometry is challenging because of the huge chemical space of co-existent glycoforms, i.e. heterogenous glycoprotein variants. Here, we report the use of an array of HPLC-MS-based approaches at different structural levels of released glycan, glycopeptide, and hitherto unexplored intact glycoforms to scrutinize the biopharmaceutical Myozyme®, containing the highly complex lysosomal enzyme recombinant acid α-glucosidase. The intrinsic heterogeneity of recombinant acid α-glucosidase glycoforms was unraveled using a novel strong anion-exchange (SAX)-HPLC-MS approach involving a pH gradient of volatile buffers to facilitate chromatographic separation of glycoforms based on their degree of sialylation followed by the acquisition of native mass spectra in an Orbitrap mass spectrometer. The large set of interdepend data acquired at different structural levels was integrated using a set of bioinformatics tools and allowed the annotation of intact glycoforms unraveling more than 1,000,000 different intact glycoform structures. Finally, for the first time, we sought to validate the intact glycoform annotations by integrating experimental data on the enzymatically dissected proteoforms revealing the strengths but also intrinsic limitations of this approach for fully characterising such highly complex glycoproteins by mass spectrometry.

The neglected tropical disease onchocerciasis, or river blindness, is caused by infection with the filarial nematode Onchocerca volvulus. Current estimates indicate that 17 million people are infected worldwide, the majority of them living in Africa. Today there are no non-invasive tests available that can detect ongoing infection, and that can be used for effective monitoring of elimination programs. In addition, to enable pharmacodynamic studies with novel macrofilaricide drug candidates, surrogate endpoints and efficacy biomarkers are needed but are non-existent. We describe the use of a multimodal untargeted mass spectrometry-based approach (metabolomics and lipidomics) to identify onchocerciasis-associated metabolites in urine and plasma, and of specific lipid features in plasma of infected individuals (O. volvulus infected cases: 68 individuals with palpable nodules; lymphatic filariasis cases: 8 individuals; non-endemic controls: 20 individuals). This work resulted in the identification of elevated concentrations of the plasma metabolites inosine and hypoxanthine as biomarkers for filarial infection, and of the urine metabolite cis-cinnamoylglycine (CCG) as biomarker for O. volvulus. During the targeted validation study, metabolite-specific cutoffs were determined (inosine: 34.2 ng/ml; hypoxanthine: 1380 ng/ml; CCG: 29.7 ng/ml) and sensitivity and specificity profiles were established. Subsequent evaluation of these biomarkers in a non-endemic population from a different geographical region invalidated the urine metabolite CCG as biomarker for O. volvulus. The plasma metabolites inosine and hypoxanthine were confirmed as biomarkers for filarial infection. With the availability of targeted LC-MS procedures, the full potential of these 2 biomarkers in macrofilaricide clinical trials, MDA efficacy surveys, and epidemiological transmission studies can be investigated., Author summary Today’s diagnosis of infection with the filarial parasite Onchocerca volvulus mainly depends on the microscopic analysis of skin biopsies and serological testing. The work presented here describes the use of multiple mass spectrometry-based screening methods (metabolomics and lipidomics) to search for biomarkers indicative of infection with Onchocerca volvulus. This resulted in the identification of elevated concentrations of the plasma metabolites inosine and hypoxanthine as biomarkers for filarial infection, and of the urine metabolite cis-cinnamoylglycine as biomarker for O. volvulus. Further evaluation of these biomarkers in a geographically distinct non-endemic population however invalidated the use of urine cis-cinnamoylglycine. These findings are of utmost importance as it not only opens new avenues in the development of non-invasive diagnostic tools for filarial infections, but also emphasizes the need for evaluation and validation of newly discovered biomarkers in different populations from different geographies.

Rising population density and global mobility are among the reasons why pathogens such as SARS-CoV-2, the virus that causes COVID-19, spread so rapidly across the globe. The policy response to such pandemics will always have to include accurate monitoring of the spread, as this provides one of the few alternatives to total lockdown. However, COVID-19 diagnosis is currently performed almost exclusively by reverse transcription polymerase chain reaction (RT-PCR). Although this is efficient, automatable, and acceptably cheap, reliance on one type of technology comes with serious caveats, as illustrated by recurring reagent and test shortages. We therefore developed an alternative diagnostic test that detects proteolytically digested SARS-CoV-2 proteins using mass spectrometry (MS). We established the Cov-MS consortium, consisting of 15 academic laboratories and several industrial partners to increase applicability, accessibility, sensitivity, and robustness of this kind of SARS-CoV-2 detection. This, in turn, gave rise to the Cov-MS Digital Incubator that allows other laboratories to join the effort, navigate, and share their optimizations and translate the assay into their clinic. As this test relies on viral proteins instead of RNA, it provides an orthogonal and complementary approach to RT-PCR using other reagents that are relatively inexpensive and widely available, as well as orthogonally skilled personnel and different instruments. Data are available via ProteomeXchange with identifier PXD022550. ispartof: JACS AU vol:1 issue:6 pages:750-765 ispartof: location:United States status: published

Fully automated characterization of monoclonal antibody (mAb) charge variants using four-dimensional liquid chromatography-mass spectrometry (4D-LC-MS) is reported and illustrated. Charge variants resolved by cation-exchange chromatography (CEX) using a salt- or pH-gradient are collected in loops installed on a multiple heart-cutting valve and consequently subjected to online desalting, denaturation, reduction and trypsin digestion prior to LC-MS based peptide mapping. This innovation which substantially reduces turnaround time, sample manipulation, loss and artefacts and increases information gathering, is described in great technical detail, and applied to characterize the charge heterogeneity associated with three therapeutic mAbs. Sequence coverages > 95% are obtained for major and minor charge variants (> 1.0%). Post-translational modifications (PTMs) and modification sites are readily revealed in a repeatable manner including unstable succinimide intermediates which are not maintained when performing classical in-solution overnight digestion of offline collected CEX peaks.

The majority of recombinant mAb products contain heterogeneous charge variants, commonly the result of post-translational modifications occurring during cell culture and accumulated during production, formulation and storage. MB02 is a biosimilar mAb to bevacizumab. Similarity data of charge variants for biosimilars against its reference products must be generated to demonstrate consistency in product quality and to ensure efficacy and safety. The goal of this work was to isolate seven charge variants of MB02 and Avastin® by semi-preparative cation exchange chromatography followed by purity test and extended analytical characterization to prove similarity. Although poor purity obtained for minor variants complicated data interpretation, an in-depth insight into the charge variants pattern of MB02 compared to Avastin® was obtained, contributing to a better understanding of modifications associated to microheterogeneity. To our knowledge, this is the first comparative analytical study of individual charge variants of a bevacizumab biosimilar following a head-to head approach and the most comprehensive N-glycosylation assessment of IgG1 charge variants. Although modifications related to N- and C-terminal, N-glycans, size heterogeneity or deamidation were specifically enriched among low abundant charge variants, they did not affect binding affinity to VEGF or FcRn and in vitro potency compared with the main species or unfractionated material.

The identification and accurate quantitation of the various glycoforms contained in therapeutic monoclonal antibodies (mAbs) is one of the main analytical needs in the biopharmaceutical industry, and glycosylation represents a crucial critical quality attribute (CQA) that needs to be addressed. Currently, the reference method for performing such identification/quantitation consists of the release of the N-glycan moieties from the mAb, their labelling with a specific dye (e.g., 2-AB or RFMS) and their analysis by HILIC-FLD or HILIC-MS. In this contribution, the potential of a new cost- and time-effective analytical approach performed at the protein subunit level (middle-up) was investigated for quantitative purposes and compared with the reference methods. The robustness of the approach was first demonstrated by performing the relative quantification of the glycoforms related to a well characterized mAb, namely adalimumab. Then, the workflow was applied to various glyco-engineered mAb products (i.e., obinutuzumab, benralizumab and atezolizumab). Finally, the glycosylation pattern of infliximab (Remicade®) was assessed and compared to two of its commercially available biosimilars (Remsima® and Inflectra®). The middle-up analysis proved to provide accurate quantitation results and has the added potential to be used as multi-attribute monitoring method.

This study describes the fully automated middle-up characterization of monoclonal antibodies (mAbs) and next-generation variants by online reduction liquid chromatography-mass spectrometry (LC-MS). Proteins were trapped on-column and subjected to online desalting, denaturation and reduction prior to reversed phase elution of the created subunits in the MS. The evaluation of more than 20 different therapeutic proteins including full length mAbs (subclasses IgG1, IgG2 and IgG4), bispecific antibodies, antibody fragments, fusion proteins and antibody-drug conjugates (ADC) revealed that the online reduction method is as powerful as the widely applied offline sample preparation with dithiothreitol (DTT) as reducing agent and guanidine hydrochloride (Gnd.HCl) as denaturant and tackles some major disadvantages associated with the latter method, i.e. corrosion of stainless steel components, adduct formation impacting spectral quality and sample stability. The value of the online reduction LC-MS method is also enforced by its ability to reveal unstable antibody variants such as succinimide intermediates of asparagine deamidation and aspartic acid isomerization which are often lost when using the offline sample preparation method. The performance of the online reduction LC-MS set-up was verified and it was revealed that the method is precise with RSD values below 0.25% and 3.0% for retention time and area, respectively. Carry-over is within acceptable limits (< 0.5%) and the reducing buffer is stable up to 24 hours.

Infections with intestinal worms, such as Ascaris lumbricoides, affect hundreds of millions of people in all tropical and subtropical regions of the world. Through large-scale deworming programs, World Health Organization aims to reduce moderate-to-heavy intensity infections below 1%. Current diagnosis and monitoring of these control programs are solely based on the detection of worm eggs in stool. Here we describe how metabolome analysis was used to identify the A. lumbricoides-specific urine biomarker 2-methyl pentanoyl carnitine (2-MPC). This biomarker was found to be 85.7% accurate in determining infection and 90.5% accurate in determining a moderate-to-heavy infection. Our results also demonstrate that there is a correlation between 2-MPC levels in urine and A. lumbricoides DNA detected in stool. Furthermore, the levels of 2-MPC in urine were shown to rapidly and strongly decrease upon administration of a standard treatment (single oral dose of 400 mg albendazole). In an Ascaris suum infection model in pigs, it was found that, although 2-MPC levels were much lower compared to humans, there was a significant association between urinary 2-MPC levels and both worm counts (p = 0.023) and the number of eggs per gram (epg) counts (p A. lumbricoides-specific biomarker that can be used to monitor infection intensity.

An introduction from guest editors, Koen Sandra and Davy Guillarme, on this special supplement from LCGC Europe focusing on recent developments in biopharmaceutical analysis.

Urinary extracellular vesicles (EVs) are an attractive source of biomarkers for urological diseases. A crucial step in biomarker discovery studies is the determination of the variation parameters to perform a sample size calculation. In this way, a biomarker discovery study with sufficient statistical power can be performed to obtain biologically significant biomarkers. Here, a variation study was performed on both the protein and lipid content of urinary EVs of healthy individuals, aged between 52 and 69 years. Ultrafiltration (UF) in combination with size exclusion chromatography (SEC) was used to isolate the EVs from urine. Different experimental variation set-ups were used in this variation study. The calculated standard deviations (SDs) of the 90% least variable peptides and lipids did not exceed 2 and 1.2, respectively. These parameters can be used in a sample size calculation for a well-designed biomarker discovery study at the cargo of EVs.

In recent years, two-dimensional liquid chromatography (2D-LC) has seen an enormous evolution and one of the fields where it is being widely adopted is in the analysis of therapeutic monoclonal antibodies (mAbs). We here further add to the many flavours of this powerful technology. Workflows based on heart-cutting (LC-LC) and comprehensive (LC × LC) 2D-LC are described that allow to guide the clone selection process in mAb and biosimilar development. Combining Protein A affinity chromatography in the first dimension with size exclusion (SEC), cation exchange (CEX) or reversed-phase liquid chromatography-mass spectrometry (RPLC–MS) in the second dimension simultaneously allows to assess mAb titer and critical structural aspects such as aggregation, fragmentation, charge heterogeneity, molecular weight (MW), amino acid sequence and glycosylation. Complementing the LC-LC measurements at intact protein level with LC × LC based peptide mapping provides the necessary information to make clear decisions on which clones to take further into development.

The aim of this study was to evaluate the practical possibilities and limitations of several recently introduced size exclusion chromatographic (SEC) columns of 150×4.6mm, sub-3μm (Agilent AdvanceBioSEC 2.7μm, Tosoh TSKgel UP-SW3000 2.0μm, Phenomenex Yarra SEC X-150 1.8μm and Waters Acquity BEH200 1.7μm) for the separation of biopharmaceutical proteins. For this purpose, some model proteins were tested, as well as several commercial therapeutic monoclonal antibodies (mAbs) and antibody-drug-conjugates (ADCs). Calibration curves were drawn to highlight the applicability of these new SEC columns for the separation of mAbs, ADCs and their aggregates, despite some differences in their nominal pore diameter (vary from 150 to 300Å). The kinetic performance (van Deemter curves and kinetic pots) was evaluated. Columns packed with 1.7-2.0μm particles improved the plate count by a factor of 1.5-2 compared to 2.7μm particles, which is in agreement with theoretical expectations. Finally, possible secondary hydrophobic and/or electrostatic interactions between the SEC stationary phases and biopharmaceutical proteins were systematically studied. Significant differences in nonspecific interactions were observed, with hydrophobic interactions generally exerting more influence than electrostatic interactions. The use of a novel bond chemistry with the AdvanceBioSEC column was found highly effective to limit non-specific interactions and pave the way to further improvements for column provider. At the end, the average resolutions achieved on the four sub-3μm SEC columns between monomer and dimer structures were comparable for ten approved mAbs products.

Monoclonal antibodies are becoming a core aspect of the pharmaceutical industry. Together with a huge therapeutic potential, these molecules come with a structural complexity that drives state-of-the-art chromatography and mass spectrometry (MS) to its limits. This article discusses the use of micro-pillar array columns in combination with mass spectrometry for peptide mapping of monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs). Micro-pillar array columns are produced by a lithographic etching process creating a perfectly ordered separation bed on a silicon chip. As a result of the order existing in these columns, peak dispersion is minimized and highly efficient peptide maps are generated, providing enormous structural detail. Using examples from the author's laboratory, the performance of these columns is illustrated.

Comprehensive two-dimensional liquid chromatography (LC×LC) is here proposed as a novel tool for peptide mapping of therapeutic monoclonal antibodies in both RD and routine (QA/QC) environments. This is illustrated by the analysis of the tryptic digest of trastuzumab (Herceptin) applying a commercially available two-dimensional 2D-LC system. Three different LC×LC combinations, i.e., strong cation-exchange × reversed-phase (SCX×RP), reversed-phase × reversed-phase (RP×RP), and hydrophilic interaction × reversed-phase (HILIC×RP), are reported. Detection was carried out using both UV detection (DAD) and mass spectrometry (MS). Several challenges related to the application of LC×LC in peptide mapping and the hyphenation to MS are addressed. The applicability of LC×LC in the assessment of identity, purity, and comparability is demonstrated by the analysis of different Herceptin innovator production batches, a Herceptin biosimilar in development and of stressed samples. The described methodology was shown to be precise in terms of peak volume and (2)D retention time opening interesting perspectives for use in QA/QC testing.

Protein biopharmaceuticals such as monoclonal antibodies and therapeutic proteins are currently in widespread use for the treatment of various life-threatening diseases including cancer, autoimmune disorders, diabetes and anemia. The complexity of protein therapeutics is far exceeding that of small molecule drugs; hence, unraveling this complexity represents an analytical challenge. The current review provides the reader with state-of-the-art chromatographic and mass spectrometric tools available to dissect primary and higher order structures, post-translational modifications, purity and impurity profiles and pharmacokinetic properties of protein therapeutics.

Here we report a metabolomics discovery study conducted on blood serum samples of patients in different stages of chronic kidney disease (CKD). Metabolites were monitored on a quality controlled holistic platform combining reversed-phase liquid chromatography coupled to high-resolution quadrupole time-of-flight mass spectrometry in both negative and positive ionization mode and gas chromatography coupled to quadrupole mass spectrometry. A substantial portion of the serum metabolome was thereby covered. Eighty-five metabolites were shown to evolve with CKD progression of which 43 metabolites were a confirmation of earlier reported uremic retention solutes and/or uremic toxins. Thirty-one unique metabolites were revealed which were increasing significantly throughout CKD progression, by a factor surpassing the level observed for creatinine, the currently used biomarker for kidney function. Additionally, 11 unique metabolites showed a decreasing trend.

The global non-targeted analysis of various biomolecules in a variety of sample sources gained momentum in recent years. Defined as the study of the full lipid complement of cells, tissues and organisms, lipidomics is currently evolving out of the shadow of the more established omics sciences including genomics, transcriptomics, proteomics and metabolomics. In analogy to the latter, lipidomics has the potential to impact on biomarker discovery, drug discovery/development and system knowledge, amongst others. The tools developed by lipid researchers in the past, complemented with the enormous advancements made in recent years in mass spectrometry and chromatography, and the implementation of sophisticated (bio)-informatics tools form the basis of current lipidomics technologies.

Psoralen and ultraviolet A light (PUNTA) are used to kill pathogens in blood products and as a treatment of aberrant cell proliferation in dermatitis, cutaneous T-cell lymphoma, and graft versus-host disease. DNA damage is well described, but the direct effects of PUVA on cell signal transduction are poorly understood. Because platelets are anucleate and contain archetypal signal transduction machinery, they are ideally suited to address this. Lipidomics on platelet membrane extracts showed that psoralen forms adducts with unsaturated carbon bonds of fatty acyls in all major phospholipid classes after PUVA. Such adducts increased lipid packing as measured by a blue shift of an environment-sensitive fluorescent probe in model liposomes. Furthermore, the interaction of these liposomes with lipid order-sensitive proteins like amphipathic lipid-packing sensor and a-synuclein was inhibited by PUVA. In platelets, PUVA caused poor membrane binding of Akt and Bruton's tyrosine kinase effectors following activation of the collagen glycoprotein VI and thrombin protease-activated receptor (PAR) 1. This resulted in defective Akt phosphorylation despite unaltered phosphatidylinositol 3,4,5-trisphosphate levels. Downstream integrin activation was furthermore affected similarly by PUVA following PAR1 (effective half-maximal concentration (EC), 8.4 +/- 1.1 versus 4.3 +/- 1.1 mu M) and glycoprotein VI (EC50, 1.61 +/- 0.85 versus 0.26 +/- 0.21 mu g/ml) but not PAR4 (EC50, 50 +/- 1 versus 58 +/- 1 mu m) signal transduction. Our findings were confirmed in T-cells ftom graft-versus-host disease patients treated with extracorporeal photopheresis, a form of systemic PUVA. In conclusion, PUVA increases the order of lipid phases by covalent modification of phospholipids, thereby inhibiting membrane recruitment of effector kinases.

Antibody-drug conjugates might be the magic bullets referred to by Paul Ehrlich over 100 years ago. Together with a huge therapeutic potential, these molecules come with a structural complexity that drives state-of-the-art chromatography and mass spectrometry to its limits. The use of multiple heart-cutting (mLC-LC) and comprehensive (LC×LC) multidimensional LC in combination with high resolution mass spectrometry for the characterization of the lysine conjugated antibody-drug conjugate ado-trastuzumab emtansine, commercialized as Kadcyla, is presented. By combining protein and peptide measurements, attributes such as drug loading, drug distribution and drug conjugation sites can be assessed in an elegant manner.

An LC-MS based method for the profiling and characterization of ceramide species in the upper layer of human skin is described. Ceramide samples, collected by tape stripping of human skin, were analyzed by reversed-phase liquid chromatography coupled to high-resolution quadrupole time-of-flight mass spectrometry operated in both positive and negative electrospray ionization mode. All known classes of ceramides could be measured in a repeatable manner. Furthermore, the data set showed several undiscovered ceramides, including a class with four hydroxyl functionalities in its sphingoid base. High-resolution MS/MS fragmentation spectra revealed that each identified ceramide species is composed of several skeletal isomers due to variation in carbon length of the respective sphingoid bases and fatty acyl building blocks. The resulting variety in skeletal isomers has not been previously demonstrated. It is estimated that over 1000 unique ceramide structures could be elucidated in human stratum corneum. Ceramide species with an even and odd number of carbon atoms in both chains were detected in all ceramide classes. Acid hydrolysis of the ceramides, followed by LC-MS analysis of the end-products, confirmed the observed distribution of both sphingoid bases and fatty acyl groups in skin ceramides. The study resulted in an accurate mass retention time library for targeted profiling of skin ceramides. It is furthermore demonstrated that targeted data processing results in an improved repeatability versus untargeted data processing (72.92% versus 62.12% of species display an RSD < 15%).

A systematic study is reported on the performance of long monolithic capillary columns in gradient mode. Using a commercial nano-LC system, reversed-phase peptide separations obtained through UV-detection were conducted. The chromatographic performance, in terms of conditional peak capacity and peak productivity, was investigated for different gradient times (varying between 90 and 1320 min) and different column lengths (0.25, 1, 2 and 4 m) all originating from a single 4 m long column. Peak capacities reaching values up to n = 103 were measured in case of the 4 m long column demonstrating the high potential of these long monoliths for the analysis of complex biological mixtures, amongst others. In addition, it was found that the different column fragments displayed similar flow resistance as well as consistent chromatographic performance in accordance with chromatographic theory indicating that the chromatographic bed of the original 4 m long column possessed a structural homogeneity over its entire length.

International audience; Molluscan hemocyanins (Hcs) have recently received particular interest due to their significant immunostimulatory properties. This is mainly related to their high carbohydrate content and specific monosaccharide composition. We have now analyzed the oligosaccharides and the carbohydrate linkage sites of the Rapana venosa hemocyanin (RvH) using different approaches. We analyzed a number of glycopeptides by LC/ESI-MS/MS and identified the sugar chains and peptide sequences of 12 glycopeptides. Additionally, the potential carbohydrate linkage sites of 2 functional units, RvH-b and RvH-c, were determined by gene sequence analysis. Only RvH-c shows a potential N-glycosylation site. During this study, we discovered a highly conserved linker-intron, separating the coding exons of RVH-b and RvH-c. Following reports on antiviral properties from arthropod hemocyanin, we conducted a preliminary study of the antiviral activity of RvH and the functional units RvH-b and RvH-c. We show that the glycosylated FU RvH-c has antiviral properties against the respiratory syncytial virus (RSV), whereas native RvH and the nonglycosylated FU RvH-b have not. This is the first report of the fact that also molluscan hemocyanin functional units possess antiviral activity.

Multidimensional liquid-based separation techniques are described for maximizing the resolution of the enormous number of peptides generated upon tryptic digestion of proteomes, and hence, reduce the spatial and temporal complexity of the sample to a level that allows successful mass spectrometric analysis. This review complements the previous contribution on unidimensional high performance liquid chromatography (HPLC). Both chromatography and electrophoresis will be discussed albeit with reversed-phase HPLC (RPLC) as the final separation dimension prior to MS analysis.

As an alternative to the classical approach of combining strong cation exchange SCX-LC and RP-LC for the separation of complex proteomic samples, this essay describes the online comprehensive RP-LCxRP-LC separation of BSA and human blood serum. High orthogonality and peak capacity are achieved through the application of a significantly different pH in the two dimensions. The coupling of fused-core columns in series ensures high efficiency in the first dimension, while a previously designed interface with parallel second dimension columns further enhances the separation capability of the comprehensive system.

The present paper introduces the use of a weak cation-exchange/crown ether column in the proteomics field. The 18-crown-6 ether functionality is well-known to selectively complex ammonium and monoalkylammonium ions, which should make this column highly suitable to trap peptides with free alpha-NH(2) or free epsilon-NH(2) groups from lysine side chains. This unique selection mechanism was put to the test in an N-teromics setup which aims for the enrichment of deliberately acetylated protein N-terminal peptides from a serum digest. It was demonstrated that peptides with free alpha-NH(2) groups and peptides with alpha-amino-acetylated groups can be separated from each other using this weak cation-exchange/crown ether column. The peptides of interest, bearing no free primary amines, were found to be significantly enriched in the column's flow through. At the same time a favorable coenrichment of N-glycosylated peptides was observed. To obtain more insight in the contributions of the two distinct column functionalities, i.e., the weak cation exchanger and the crown ether, the experimental data were checked against a theoretical prediction of the outcome.

Sample complexity and dynamic range constitute enormous challenges in proteome analysis. The back-end technology in typical proteomics platforms, namely mass spectrometry (MS), can only tolerate a certain complexity, has a limited dynamic range per spectrum and is very sensitive towards ion suppression. Therefore, component overlap has to be minimized for successful mass spectrometric analysis and subsequent protein identification and quantification. The present review describes the advances that have been made in liquid-based separation techniques with focus on the recent developments to boost the resolving power. The review is divided in two parts; the first part deals with unidimensional liquid chromatography and the second part with bi- and multidimensional liquid-based separation techniques. Part 1 mainly focuses on reversed-phase HPLC due to the fact that it is and will, in the near future, remain the technique of choice to be hyphenated with MS. The impact of increasing the column length, decreasing the particle diameter, replacing the traditional packed beds by monolithics, amongst others, is described. The review is complemented with data obtained in the laboratories of the authors.

The previously reported COmbined FRActional DIagonal Chromatography (COFRA-DIC) methodology, in which a subset of peptides representative for their parent proteins are sorted, is particularly powerful for whole proteome analysis. This peptide-centric technology is built around diagonal chromatography, where peptide separations are crucial. This paper presents high efficiency peptide separations, in which four 250 x 2.1 mm, 5 microm Zorbax 300SB-C18 columns (total length 1 m) were coupled at operating temperatures of 60'C using a dedicated LC oven and conventional LC equipment. The high efficiency separations were combined with the COFRADIC procedure. This extremely powerful combination resulted, for the analysis of serum, in an increase in the uniquely identified peptide sequences by a factor of 2.6, compared to the COFRADIC procedure on a 25 cm column. This is a reflection of the increased peak capacity obtained on the 1 m column, which was calculated to be a factor 2.7 higher than on the 25 cm column. Besides more efficient sorting, less ion suppression was noticed.

Micro-vial pyrolysis (PyroVial) was used to study the production of compounds important for the aroma of heat-treated natural products such as tobacco. Firstly, a mixture of glucose and proline was pyrolyzed as model, as this sugar and amino acid are also abundant in tobacco leaf (Nicotiana tobacum L.). The pyrolysate was analyzed using headspace-GC–MS, liquid injection GC–MS and LC–MS. Next, micro-vial pyrolysis in combination with LC–MS was applied to tobacco leaf extract. Using MS deconvolution, molecular feature extraction and differential analysis it was possible to identify Amadori intermediates of the Maillard reaction in the tobacco leaf extract. The intermediate disappeared as was the case for 1-deoxy-1-prolino-β-d-fructose or the concentration decreased in the pyrolysate compared to the original extract such as for the 1-deoxy-1-[2-(3-pyridyl)-1-pyrrolidinyl]-β-d-fructose isomers indicating that Amadori intermediates are important precursors for aroma compound formation.

Induced lung sputum is a valuable matrix in the study of respiratory diseases. Although the methodology of sputum collection has evolved to a point where it is repeatable and responsive to inflammation, its use in molecular profiling studies is still limited. Here, an in-depth lipid profiling of induced lung sputum using high-resolution liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-QTOF MS) is described. An enormous complexity in lipid composition could be revealed. Over 1500 intact lipids, originating from 6 major lipid classes, have been accurately identified in 120 tit of induced sputum. By number and measured intensity, glycerophospholipids represent the largest lipid class, followed by sphingolipids, glycerolipids, fatty acyls, sterol lipids, and prenol lipids. Several prenol lipids, originating from tobacco, could be detected in the lung sputum of smokers. To illustrate the utility of the methodology in studying respiratory diseases, a comparative lipid screening was performed on lung sputum extracts in order to study the effect of Chronic Obstructive Pulmonary Disease (COPD) on the lung barrier lipidome. Results show that sphingolipid expression in induced sputum significantly differs between smokers with and without COPD.

N-Glycosylation of cellobiohydrolase I from the fungus Trichoderma reesei (strain Rut-C30) is studied using a combination of electrophoretic, chromatographic and mass spectrometric techniques. As four potential N-glycosylation sites and several uncharged and phosphorylated high-mannose glycans are present, a large number of glycoforms and phospho-isoforms can be expected. Isoelectric focusing both in gel and in capillary format was successfully applied for the separation of the phospho-isoforms. They were extracted in their intact form from the gel and subsequently analysed by nanospray-Q-TOF-MS, thereby making use of a powerful two-dimensional technique. Nano-LC/MS/MS on a Q-Trap MS further allowed the determination of the glycosylation sites. As a novel approach, an oxonium ion was used in precursor ion scanning for selective detection of glycopeptides containing phosphorylated high-mannose glycans.

The glycosylation of Cel7A (CBH I) from Trichoderma reeseivaries considerably when the fungus is grown under differentconditions. As shown by ESI-MS and PAG-IEF analyses ofboth intact protein and the isolated catalytic core module, themicroheterogeneity originates mainly from the variable ratioof single N-acetylglucosamine over high-mannose structureson the three N-glycosylation sites and from the presence orabsence of phosphate residues. Fully N- and O-glycosylatedCel7A can only be isolated from minimal medium and prob-ablyreflectstheinitialcomplexityoftheproteinonleavingtheglycosynthetic pathway. Extracellular activities are responsi-ble for postsecretorial modifications in other cultivation con-ditions: a-(1!2)-mannosidase, a-(1!3)-glucosidase and anEndo H type activity participate in N-deglycosylation (core),whereas a phosphatase and a mannosidase are probablyresponsible for hydrolysis of O-glycans (linker). The effectsare most prominent in corn steep liquor–enriched media,where the pH is closer to the pH optimum (5–6) of theseextracellular hydrolases. In minimal medium, the low pH andthepresenceofproteasescouldexplainfortheabsenceofsuchactivities. On the other hand, phosphodiester linkages in thecatalytic module are only observed under specific conditions.The extracellular trigger is still unknown, but manno-phosphorylationmayberegulatedintracellularlybya-(1!2)-mannosidases and phosphomannosyl transferases competingfor the same intermediate in the glycosynthetic pathway.Key words: Cel7A/endoglycosidase/N- andO-glycosylation/postsecretorial modifications/Trichoderma reeseiIntroductionCellulases belonging to different glycosyl hydrolase familiesvery often exhibit a multidomain structure (Coutinhoand Henrissat, 1999): a catalytic domain (core) and acarbohydrate-binding module are separated by a linkerpeptide rich in proline, serine, and threonine. Fungalcellulases carry posttranslational modifications on bothdomains: Whereas the linker peptide is highly O-glycosylated, N-glycosylation seems to be restricted to thecore. Filamentous fungi typically synthesize short-chain O-and N-glycans, resembling the mammalian high-mannosetype rather than the hyperglycosylated yeast structures.The glycosylation of cellobiohydrolase I (CBH I, Cel7A),a cellulase abundantly expressed by most Trichodermareesei strains, has been studied particularly well(Table I). Trichoderma cellulases appear in several isoformswith similar catalytic and adsorption properties (Medveet al., 1998), and it has been shown that both N- andO-glycans account for the many isoforms of Cel7A(Pakula et al., 2000).Detailed structural investigations (Harrison et al., 1998;Klarskov et al., 1997) revealed the N-glycosylation inT. reesei Cel7A from respectively strains QM9414 andALKO2877 (derived from QM9414): Single GlcNAc resi-dues in three (Asn45, Asn270, and Asn384) out of fourpotential sites were characterized in the catalytic domain.More complex N-glycan structures were observed withCel7A from the hyperproducing mutant Rut-C30 strain(Maras et al., 1997). The majority are monoglucosylatedhigh-mannose glycans (GlcMan