Proteome Dynamics During Transition From Exponential to Stationary Phase Under Aerobic and Anaerobic Conditions in Yeast.

The yeast Saccharomyces cerevisiae is a widely-used eukaryotic model organism and a promising cell factory for industry. However, despite decades of research, the regulation of its metabolism is not yet fully understood, and its complexity represents a major challenge for engineering and optimizing biosynthetic routes. Recent studies have demonstrated the potential of resource and proteomic allocation data in enhancing models for metabolic processes. However, comprehensive and accurate proteome dynamics data that can be used for such approaches are still very limited. Therefore, we performed a quantitative proteome dynamics study to comprehensively cover the transition from exponential to stationary phase for both aerobically and anaerobically grown yeast cells. The combination of highly controlled reactor experiments, biological replicates, and standardized sample preparation procedures ensured reproducibility and accuracy. In addition, we selected the CEN.PK lineage for our experiments because of its relevance for both fundamental and applied research. Together with the prototrophic standard haploid strain CEN.PK113-7D, we also investigated an engineered strain with genetic minimization of the glycolytic pathway, resulting in the quantitative assessment of 54 proteomes. The anaerobic cultures showed remarkably less proteome-level changes compared with the aerobic cultures, during transition from the exponential to the stationary phase as a consequence of the lack of the diauxic shift in the absence of oxygen. These results support the notion that anaerobically growing cells lack resources to adequately adapt to starvation. This proteome dynamics study constitutes an important step toward better understanding of the impact of glucose exhaustion and oxygen on the complex proteome allocation process in yeast. Finally, the established proteome dynamics data provide a valuable resource for the development of resource allocation models as well as for metabolic engineering efforts.

Simple Routes to Stable Isotope-Coded Native Glycans.

Understanding the biological role of protein-linked glycans requires the reliable identification of glycans. Isomer separation and characterization often entail mass spectrometric detection preceded by high-performance chromatography on porous graphitic carbon. To this end, stable isotope-labeled glycans have emerged as powerful tools for retention time normalization. Hitherto, such standards were obtained by chemoenzymatic or purely enzymatic methods, which introduce, e.g., 13C-containing N-acetyl groups or galactose into native glycans. Glycan release with anhydrous hydrazine opens another route for heavy isotope introduction via concomitant de-N-acetylation. Here, we describe that de-N-acetylation can also be achieved with hydrazine hydrate, which is a more affordable and less hazardous reagent. Despite the slower reaction rate, complete conversion is achievable in 72 h at 100 °C for glycans with biantennary glycans with or without sialic acids. Shorter incubation times allow for the isolation of intermediate products with a defined degree of free amino groups, facilitating introduction of different numbers of heavy isotopes. Mass encoded glycans obtained by this versatile approach can serve a broad range of applications, e.g., as internal standards for isomer-specific studies of N-glycans, O-glycans, and human milk oligosaccharide by LC-MS on either porous graphitic carbon or─following permethylation─on reversed phase.

Experimental characterization and prediction of Escherichia coli host cell proteome retention during preparative chromatography.

Purification of recombinantly produced biopharmaceuticals involves removal of host cell material, such as host cell proteins (HCPs). For lysates of the common expression host Escherichia coli (E. coli) over 1500 unique proteins can be identified. Currently, understanding the behavior of individual HCPs for purification operations, such as preparative chromatography, is limited. Therefore, we aim to elucidate the elution behavior of individual HCPs from E. coli strain BLR(DE3) during chromatography. Understanding this complex mixture and knowing the chromatographic behavior of each individual HCP improves the ability for rational purification process design. Specifically, linear gradient experiments were performed using ion exchange (IEX) and hydrophobic interaction chromatography, coupled with mass spectrometry-based proteomics to map the retention of individual HCPs. We combined knowledge of protein location, function, and interaction available in literature to identify trends in elution behavior. Additionally, quantitative structure-property relationship models were trained relating the protein 3D structure to elution behavior during IEX. For the complete data set a model with a cross-validated R2 of 0.55 was constructed, that could be improved to a R2 of 0.70 by considering only monomeric proteins. Ultimately this study is a significant step toward greater process understanding.

The metal cofactor: stationary or mobile?

Metal cofactors are essential for catalysis and enable countless conversions in nature. Interestingly, the metal cofactor is not always static but mobile with movements of more than 4 Å. These movements of the metal can have different functions. In the case of the xylose isomerase and medium-chain dehydrogenases, it clearly serves a catalytic purpose. The metal cofactor moves during substrate activation and even during the catalytic turnover. On the other hand, in class II aldolases, the enzymes display resting states and active states depending on the movement of the catalytic metal cofactor. This movement is caused by substrate docking, causing the metal cofactor to take the position essential for catalysis. As these metal movements are found in structurally and mechanistically unrelated enzymes, it has to be expected that this metal movement is more common than currently perceived. KEY POINTS: • Metal ions are essential cofactors that can move during catalysis. • In class II aldolases, the metal cofactors can reside in a resting state and an active state. • In MDR, the movement of the metal cofactor is essential for substrate docking.

Introduction of a human- and keyboard-friendly N-glycan nomenclature.

In the beginning was the word. But there were no words for N-glycans, at least, no simple words. Next to chemical formulas, the IUPAC code can be regarded as the best, most reliable and yet immediately comprehensible annotation of oligosaccharide structures of any type from any source. When it comes to N-glycans, the venerable IUPAC code has, however, been widely supplanted by highly simplified terms for N-glycans that count the number of antennae or certain components such as galactoses, sialic acids and fucoses and give only limited room for exact structure description. The highly illustrative - and fortunately now standardized - cartoon depictions gained much ground during the last years. By their very nature, cartoons can neither be written nor spoken. The underlying machine codes (e.g., GlycoCT, WURCS) are definitely not intended for direct use in human communication. So, one might feel the need for a simple, yet intelligible and precise system for alphanumeric descriptions of the hundreds and thousands of N-glycan structures. Here, we present a system that describes N-glycans by defining their terminal elements. To minimize redundancy and length of terms, the common elements of N-glycans are taken as granted. The preset reading order facilitates definition of positional isomers. The combination with elements of the condensed IUPAC code allows to describe even rather complex structural elements. Thus, this "proglycan" coding could be the missing link between drawn structures and software-oriented representations of N-glycan structures. On top, it may greatly facilitate keyboard-based mining for glycan substructures in glycan repositories.

Using artificial neural networks to accelerate flowsheet optimization for downstream process development.

An optimal purification process for biopharmaceutical products is important to meet strict safety regulations, and for economic benefits. To find the global optimum, it is desirable to screen the overall design space. Advanced model-based approaches enable to screen a broad range of the design-space, in contrast to traditional statistical or heuristic-based approaches. Though, chromatographic mechanistic modeling (MM), one of the advanced model-based approaches, can be speed-limiting for flowsheet optimization, which evaluates every purification possibility (e.g., type and order of purification techniques, and their operating conditions). Therefore, we propose to use artificial neural networks (ANNs) during global optimization to select the most optimal flowsheets. So, the number of flowsheets for final local optimization is reduced and consequently the overall optimization time. Employing ANNs during global optimization proved to reduce the number of flowsheets from 15 to only 3. From these three, one flowsheet was optimized locally and similar final results were found when using the global outcome of either the ANN or MM as starting condition. Moreover, the overall flowsheet optimization time was reduced by 50% when using ANNs during global optimization. This approach accelerates the early purification process design; moreover, it is generic, flexible, and regardless of sample material's type.

Enrichment and application of extracellular nonulosonic acids containing polymers of Accumulibacter.

Pseudaminic and legionaminic acids are a subgroup of nonulosonic acids (NulOs) unique to bacterial species. There is a lack of advances in the study of these NulOs due to their complex synthesis and production. Recently, it was seen that "Candidatus Accumulibacter" can produce Pse or Leg analogues as part of its extracellular polymeric substances (EPS). In order to employ a "Ca. Accumulibacter" enrichment as production platform for bacterial sialic acids, it is necessary to determine which fractions of the EPS of "Ca. Accumulibacter" contain NulOs and how to enrich and/or isolate them. We extracted the EPS from granules enriched with "Ca. Accumulibcater" and used size-exclusion chromatography (SEC) to separate them into different molecular weight (MW) fractions. This separation resulted in two high molecular weight (> 5500 kDa) fractions dominated by polysaccharides, with a NulO content up to 4 times higher than the extracted EPS. This suggests that NulOs in "Ca. Accumulibacter" are likely located in high molecular weight polysaccharides. Additionally, it was seen that the extracted EPS and the NulO-rich fractions can bind and neutralize histones. This opens the possibility of EPS and NulO-rich fractions as potential source for sepsis treatment drugs. KEY POINTS: • NulOs in "Ca. Accumulibacter" are likely located in high MW polysaccharides • SEC allows to obtain high MW polysaccharide-rich fractions enriched with NulOs • EPS and the NulOs-rich fractions are a potential source for sepsis treatment drugs.

Turnover of the extracellular polymeric matrix of granules performing biological phosphate removal.

Polyphosphate accumulating organisms (PAOs) are responsible for enhanced biological phosphate removal (EBPR) from wastewater, where they grow embedded in a matrix of extracellular polymeric substances (EPS). EPSs comprise a mixture of biopolymers like polysaccharides or (glyco)proteins. Despite previous studies, little is known about the dynamics of EPS in mixed cultures, and their production by PAOs and potential consumption by flanking microbes. EPSs are biodegradable and have been suggested to be a substrate for other organisms in the community. Studying EPS turnover can help elucidate their biosynthesis and biodegradation cycles. We analyzed the turnover of proteins and polysaccharides in the EPS of an enrichment culture of PAOs relative to the turnover of internal proteins. An anaerobic-aerobic sequencing batch reactor (SBR) simulating EBPR conditions was operated to enrich for PAOs. After achieving a stable culture, carbon source was switched to uniformly 13C-labeled acetate. Samples were collected at the end of each aerobic phase. EPSs were extracted by alkaline treatment. 13C enrichment in proteins and sugars (after hydrolysis of polysaccharides) in the extracted EPS were measured by mass spectrometry. The average turnover rate of sugars and proteins (0.167 and 0.192 d-1 respectively) was higher than the expected value based on the solid removal rate (0.132 d-1), and no significant difference was observed between intracellular and extracellular proteins. This indicates that EPS from the PAO enriched community is not selectively degraded by flanking populations under stable EBPR process conditions. Instead, we observed general decay of biomass, which corresponds to a value of 0.048 d-1. KEY POINTS: • Proteins showed a higher turnover rate than carbohydrates. • Turnover of EPS was similar to the turnover of intracellular proteins. • EPS is not preferentially consumed by flanking populations.

Modular, synthetic chromosomes as new tools for large scale engineering of metabolism.

The construction of powerful cell factories requires intensive genetic engineering for the addition of new functionalities and the remodeling of native pathways and processes. The present study demonstrates the feasibility of extensive genome reprogramming using modular, specialized de novo-assembled neochromosomes in yeast. The in vivo assembly of linear and circular neochromosomes, carrying 20 native and 21 heterologous genes, enabled the first de novo production in a microbial cell factory of anthocyanins, plant compounds with a broad range of pharmacological properties. Turned into exclusive expression platforms for heterologous and essential metabolic routes, the neochromosomes mimic native chromosomes regarding mitotic and genetic stability, copy number, harmlessness for the host and editability by CRISPR/Cas9. This study paves the way for future microbial cell factories with modular genomes in which core metabolic networks, localized on satellite, specialized neochromosomes can be swapped for alternative configurations and serve as landing pads for the addition of functionalities.

The pentose phosphate pathway of cellulolytic clostridia relies on 6-phosphofructokinase instead of transaldolase.

The genomes of most cellulolytic clostridia do not contain genes annotated as transaldolase. Therefore, for assimilating pentose sugars or for generating C5 precursors (such as ribose) during growth on other (non-C5) substrates, they must possess a pathway that connects pentose metabolism with the rest of metabolism. Here we provide evidence that for this connection cellulolytic clostridia rely on the sedoheptulose 1,7-bisphosphate (SBP) pathway, using pyrophosphate-dependent phosphofructokinase (PPi-PFK) instead of transaldolase. In this reversible pathway, PFK converts sedoheptulose 7-phosphate (S7P) to SBP, after which fructose-bisphosphate aldolase cleaves SBP into dihydroxyacetone phosphate and erythrose 4-phosphate. We show that PPi-PFKs of Clostridium thermosuccinogenes and Clostridium thermocellum indeed can convert S7P to SBP, and have similar affinities for S7P and the canonical substrate fructose 6-phosphate (F6P). By contrast, (ATP-dependent) PfkA of Escherichia coli, which does rely on transaldolase, had a very poor affinity for S7P. This indicates that the PPi-PFK of cellulolytic clostridia has evolved the use of S7P. We further show that C. thermosuccinogenes contains a significant SBP pool, an unusual metabolite that is elevated during growth on xylose, demonstrating its relevance for pentose assimilation. Last, we demonstrate that a second PFK of C. thermosuccinogenes that operates with ATP and GTP exhibits unusual kinetics toward F6P, as it appears to have an extremely high degree of cooperative binding, resulting in a virtual on/off switch for substrate concentrations near its K½ value. In summary, our results confirm the existence of an SBP pathway for pentose assimilation in cellulolytic clostridia.

Natranaerofaba carboxydovora gen. nov., sp. nov., an extremely haloalkaliphilic CO-utilizing acetogen from a hypersaline soda lake representing a novel deep phylogenetic lineage in the class 'Natranaerobiia'.

An anaerobic enrichment with CO from sediments of hypersaline soda lakes resulted in a methane-forming binary culture, whereby CO was utilized by a bacterium and not the methanogenic partner. The bacterial isolate ANCO1 forms a deep-branching phylogenetic lineage at the level of a new family within the class 'Natranaerobiia'. It is an extreme haloalkaliphilic and moderate thermophilic acetogen utilizing CO, formate, pyruvate and lactate as electron donors and thiosulfate, nitrate (reduced to ammonia) and fumarate as electron acceptors. The genome of ANCO1 encodes a full Wood-Ljungdahl pathway allowing for CO oxidation and acetogenic conversion of pyruvate. A locus encoding Nap nitrate reductase/NrfA ammonifying nitrite reductase is also present. Thiosulfate respiration is encoded by a Phs/Psr-like operon. The organism obviously relies on Na-based bioenergetics, since the genome encodes for the Na+ -Rnf complex, Na+ -F1F0 ATPase and Na+ -translocating decarboxylase. Glycine betaine serves as a compatible solute. ANCO1 has an unusual membrane polar lipid composition dominated by diethers, more common among archaea, probably a result of adaptation to multiple extremophilic conditions. Overall, ANCO1 represents a unique example of a triple extremophilic CO-oxidizing anaerobe and is classified as a novel genus and species Natranaerofaba carboxydovora in a novel family Natranaerofabacea.

Bisecting Lewis X in Hybrid-Type N-Glycans of Human Brain Revealed by Deep Structural Glycomics.

The importance of protein glycosylation in the biomedical field requires methods that not only quantitate structures by their monosaccharide composition, but also resolve and identify the many isomers expressed by mammalian cells. The art of unambiguous identification of isomeric structures in complex mixtures, however, did not yet catch up with the fast pace of advance of high-throughput glycomics. Here, we present a strategy for deducing structures with the help of a deci-minute accurate retention time library for porous graphitic carbon chromatography with mass spectrometric detection. We implemented the concept for the fundamental N-glycan type consisting of five hexoses, four N-acetylhexosamines and one fucose residue. Nearly all of the 40 biosynthetized isomers occupied unique elution positions. This result demonstrates the unique isomer selectivity of porous graphitic carbon. With the help of a rather tightly spaced grid of isotope-labeled internal N-glycan, standard retention times were transposed to a standard chromatogram. Application of this approach to animal and human brain N-glycans immediately identified the majority of structures as being of the bisected type. Most notably, it exposed hybrid-type glycans with galactosylated and even Lewis X containing bisected N-acetylglucosamine, which have not yet been discovered in a natural source. Thus, the time grid approach implemented herein facilitated discovery of the still missing pieces of the N-glycome in our most noble organ and suggests itself─in conjunction with collision induced dissociation─as a starting point for the overdue development of isomer-specific deep structural glycomics.

Production of nonulosonic acids in the extracellular polymeric substances of "Candidatus Accumulibacter phosphatis".

Nonulosonic acids (NulOs) are a family of acidic carbohydrates with a nine-carbon backbone, which include different related structures, such as sialic acids. They have mainly been studied for their relevance in animal cells and pathogenic bacteria. Recently, sialic acids have been discovered as an important compound in the extracellular matrix of virtually all microbial life and in "Candidatus Accumulibacter phosphatis", a well-studied polyphosphate-accumulating organism, in particular. Here, bioaggregates highly enriched with these bacteria (approx. 95% based on proteomic data) were used to study the production of NulOs in an enrichment of this microorganism. Fluorescence lectin-binding analysis, enzymatic quantification, and mass spectrometry were used to analyze the different NulOs present, showing a wide distribution and variety of these carbohydrates, such as sialic acids and bacterial NulOs, in the bioaggregates. Phylogenetic analysis confirmed the potential of "Ca. Accumulibacter" to produce different types of NulOs. Proteomic analysis showed the ability of "Ca. Accumulibacter" to reutilize and reincorporate these carbohydrates. This investigation points out the importance of diverse NulOs in non-pathogenic bacteria, which are normally overlooked. Sialic acids and other NulOs should be further investigated for their role in the ecology of "Ca. Accumulibacter" in particular, and biofilms in general. KEY POINTS: •"Ca. Accumulibacter" has the potential to produce a range of nonulosonic acids. •Mass spectrometry and lectin binding can reveal the presence and location of nonulosonic acids. •The role of nonulosonic acid in non-pathogenic bacteria needs to be studied in detail.

Shot-gun proteomics: why thousands of unidentified signals matter.

Mass spectrometry-based proteomics has become a constitutional part of the multi-omics toolbox in yeast research, advancing fundamental knowledge of molecular processes and guiding decisions in strain and product developmental pipelines. Nevertheless, post-translational protein modifications (PTMs) continue to challenge the field of proteomics. PTMs are not directly encoded in the genome; therefore, they require a sensitive analysis of the proteome itself. In yeast, the relevance of post-translational regulators has already been established, such as for phosphorylation, which can directly affect the reaction rates of metabolic enzymes. Whereas, the selective analysis of single modifications has become a broadly employed technique, the sensitive analysis of a comprehensive set of modifications still remains a challenge. At the same time, a large number of fragmentation spectra in a typical shot-gun proteomics experiment remain unidentified. It has been estimated that a good proportion of those unidentified spectra originates from unexpected modifications or natural peptide variants. In this review, recent advancements in microbial proteomics for unrestricted protein modification discovery are reviewed, and recent research integrating this additional layer of information to elucidate protein interaction and regulation in yeast is briefly discussed.

Decorating the Anammox House: Sialic Acids and Sulfated Glycosaminoglycans in the Extracellular Polymeric Substances of Anammox Granular Sludge.

Anammox (anaerobic ammonium oxidation) bacteria are important for the nitrogen cycle in both natural environments and wastewater treatment plants. These bacteria have a strong tendency to grow in aggregates like biofilms and granular sludge. To understand the formation of anammox aggregates, it is required to unravel the composition of the extracellular polymeric substances (EPS), which are produced by the bacteria to develop into aggregates and granules. Here, we investigated anionic polymers in anammox granular sludge, focussing on sialic acids and sulfated glycosaminoglycans. Quantification assays and fluorescent stains indicated that sialic acids and sulfated glycosaminoglycans were present in the anammox EPS (1.6% equivalents of sialic acids and 2.4% equivalents of sulfated glycosaminoglycans). Additionally, the potential genes for the biosynthesis of sialic acids and sulfated glycosaminoglycans were analyzed in the anammox draft genomes. The finding of these components in anammox granular sludge and previously in other nonpathogenic bacteria pointed out that sialic acids and sulfated glycosaminoglycans are worth investigating in the context of a broader function in microbial communities and biofilm systems in general.

Functional expression of a bacterial α-ketoglutarate dehydrogenase in the cytosol of Saccharomyces cerevisiae.

Efficient production of fuels and chemicals by metabolically engineered micro-organisms requires availability of precursor molecules for product pathways. In eukaryotic cell factories, heterologous product pathways are usually expressed in the cytosol, which may limit availability of precursors that are generated in other cellular compartments. In Saccharomyces cerevisiae, synthesis of the precursor molecule succinyl-Coenzyme A is confined to the mitochondrial matrix. To enable cytosolic synthesis of succinyl-CoA, we expressed the structural genes for all three subunits of the Escherichia coli α-ketoglutarate dehydrogenase (αKGDH) complex in S. cerevisiae. The E. coli lipoic-acid scavenging enzyme was co-expressed to enable cytosolic lipoylation of the αKGDH complex, which is required for its enzymatic activity. Size-exclusion chromatography and mass spectrometry indicated that the heterologously expressed αKGDH complex contained all subunits and that its size was the same as in E. coli. Functional expression of the heterologous complex was evident from increased αKGDH activity in the cytosolic fraction of yeast cell homogenates. In vivo cytosolic activity of the αKGDH complex was tested by constructing a reporter strain in which the essential metabolite 5-aminolevulinic acid could only be synthetized from cytosolic, and not mitochondrial, succinyl-CoA. To this end HEM1, which encodes the succinyl-CoA-converting mitochondrial enzyme 5-aminolevulinic acid (ALA) synthase, was deleted and a bacterial ALA synthase was expressed in the cytosol. In the resulting strain, complementation of ALA auxotrophy depended on activation of the αKGDH complex by lipoic acid addition. Functional expression of a bacterial αKGDH complex in yeast represents a vital step towards efficient yeast-based production of compounds such as 1,4-butanediol and 4-aminobutyrate, whose product pathways use succinyl-CoA as a precursor.

Identification of Glycoproteins Isolated from Extracellular Polymeric Substances of Full-Scale Anammox Granular Sludge.

ANaerobic AMMonium OXidation (anammox) is an established process for efficient nitrogen removal from wastewater, relying on anammox bacteria to form stable biofilms or granules. To understand the formation, structure, and stability of anammox granules, it is important to determine the composition of the extracellular polymeric substances (EPS). The aim of this research was to elucidate the nature of the proteins, which are the major fraction of the EPS and were suspected to be glycosylated. EPS were extracted from full-scale anammox granular sludge, dominated by " Candidatus Brocadia", and subjected to denaturing polyacrylamide gel electrophoresis. By further analysis with mass spectrometry, a high abundant glycoprotein, carrying a heterogeneous O-glycan structure, was identified. The potential glycosylation sequence motif was identical to that proposed for the surface layer protein of " Candidatus Kuenenia stuttgartiensis". The heavily glycosylated protein forms a large fraction of the EPS and was also located by lectin staining. Therefore, we hypothesize an important role of glycoproteins in the structuring of anammox granules, comparable to the importance of glycans in the extracellular matrix of multicellular organisms. Furthermore, different glycoconjugates may have distinct roles in the matrix of granular sludge, which requires more in-depth characterization of different glycoconjugates in future EPS studies.

Microarray-based MALDI-TOF mass spectrometry enables monitoring of monoclonal antibody production in batch and perfusion cell cultures.

Cell culture process monitoring in monoclonal antibody (mAb) production is essential for efficient process development and process optimization. Currently employed online, at line and offline methods for monitoring productivity as well as process reproducibility have their individual strengths and limitations. Here, we describe a matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)-based on a microarray for mass spectrometry (MAMS) technology to rapidly monitor a broad panel of analytes, including metabolites and proteins directly from the unpurified cell supernatant or from host cell culture lysates. The antibody titer is determined from the intact antibody mass spectra signal intensity relative to an internal protein standard spiked into the supernatant. The method allows a semi-quantitative determination of light and heavy chains. Intracellular mass profiles for metabolites and proteins can be used to track cellular growth and cell productivity.

A Microarray-Matrix-assisted Laser Desorption/Ionization-Mass Spectrometry Approach for Site-specific Protein N-glycosylation Analysis, as Demonstrated for Human Serum Immunoglobulin M (IgM).

We demonstrate a new approach for the site-specific identification and characterization of protein N-glycosylation. It is based on a nano-liquid chromatography microarray-matrix assisted laser desorption/ionization-MS platform, which employs droplet microfluidics for on-plate nanoliter reactions. A chromatographic separation of a proteolytic digest is deposited at a high frequency on the microarray. In this way, a short separation run is archived into thousands of nanoliter reaction cavities, and chromatographic peaks are spread over multiple array spots. After fractionation, each other spot is treated with PNGaseF to generate two correlated traces within one run, one with treated spots where glycans are enzymatically released from the peptides, and one containing the intact glycopeptides. Mining for distinct glycosites is performed by searching for the predicted deglycosylated peptides in the treated trace. An identified peptide then leads directly to the position of the "intact" glycopeptide clusters, which are located in the adjacent spots. Furthermore, the deglycosylated peptide can be sequenced efficiently in a simple collision-induced dissociation-MS experiment. We applied the microarray approach to a detailed site-specific glycosylation analysis of human serum IgM. By scanning the treated spots with low-resolution matrix assisted laser desorption/ionization-time-of-flight-MS, we observed all five deglycosylated peptides, including the one originating from the secretory chain. A detailed glycopeptide characterization was then accomplished on the adjacent, untreated spots with high mass resolution and high mass accuracy using a matrix assisted laser desorption ionization-Fourier transform-MS. We present the first detailed and comprehensive mass spectrometric analysis on the glycopeptide level for human polyclonal IgM with high mass accuracy. Besides complex type glycans on Asn 395, 332, 171, and on the J chain, we observed oligomannosidic glycans on Asn 563, Asn 402 and minor amounts of oligomannosidic glycans on the glycosite Asn 171. Furthermore, hybrid type glycans were found on Asn 402, Asn 171 and in traces Asn 332.

Differential Isotope Labeling of Glycopeptides for Accurate Determination of Differences in Site-Specific Glycosylation.

We introduce a stable isotope labeling approach for glycopeptides that allows a specific glycosylation site in a protein to be quantitatively evaluated using mass spectrometry. Succinic anhydride is used to specifically label primary amino groups of the peptide portion of the glycopeptides. The heavy form (D4(13)C4) provides an 8 Da mass increment over the light natural form (H4(12)C4), allowing simultaneous analysis and direct comparison of two glycopeptide profiles in a single MS scan. We have optimized a protocol for an in-solution trypsin digestion, a one-pot labeling procedure, and a post-labeling solid-phase extraction to obtain purified and labeled glycopeptides. We provide the first demonstration of this approach by comparing IgG1 Fc glycopeptides from polyclonal IgG samples with respect to their galactosylation and sialylation patterns using MALDI MS and LC-ESI-MS.