Autoimmune hepatitis displays distinctively high multi-antennary sialylation on plasma N-glycans compared to other liver diseases.

BACKGROUND: Changes in plasma protein glycosylation are known to functionally affect proteins and to associate with liver diseases, including cirrhosis and hepatocellular carcinoma. Autoimmune hepatitis (AIH) is a liver disease characterized by liver inflammation and raised serum levels of IgG, and is difficult to distinguish from other liver diseases. The aim of this study was to examine plasma and IgG-specific N-glycosylation in AIH and compare it with healthy controls and other liver diseases. METHODS: In this cross-sectional cohort study, total plasma N-glycosylation and IgG Fc glycosylation analysis was performed by mass spectrometry for 66 AIH patients, 60 age- and sex-matched healthy controls, 31 primary biliary cholangitis patients, 10 primary sclerosing cholangitis patients, 30 non-alcoholic fatty liver disease patients and 74 patients with viral or alcoholic hepatitis. A total of 121 glycans were quantified per individual. Associations between glycosylation traits and AIH were investigated as compared to healthy controls and other liver diseases. RESULTS: Glycan traits bisection (OR: 3.78 [1.88-9.35], p-value: 5.88 × 10- 3), tetraantennary sialylation per galactose (A4GS) (OR: 2.88 [1.75-5.16], p-value: 1.63 × 10- 3), IgG1 galactosylation (OR: 0.35 [0.2-0.58], p-value: 3.47 × 10- 5) and hybrid type glycans (OR: 2.73 [1.67-4.89], p-value: 2.31 × 10- 3) were found as discriminators between AIH and healthy controls. High A4GS differentiated AIH from other liver diseases, while bisection associated with cirrhosis severity. CONCLUSIONS: Compared to other liver diseases, AIH shows distinctively high A4GS levels in plasma, with potential implications on glycoprotein function and clearance. Plasma-derived glycosylation has potential to be used as a diagnostic marker for AIH in the future. This may alleviate the need for a liver biopsy at diagnosis. Glycosidic changes should be investigated further in longitudinal studies and may be used for diagnostic and monitoring purposes in the future.

CslA and GlxA from Streptomyces lividans form a functional cellulose synthase complex.

Filamentous growth of streptomycetes coincides with the synthesis and deposition of an uncharacterized protective glucan at hyphal tips. Synthesis of this glucan depends on the integral membrane protein CslA and the radical copper oxidase GlxA, which are part of a presumably large multiprotein complex operating at growing tips. Here, we show that CslA and GlxA interact by forming a protein complex that is sufficient to synthesize cellulose in vitro. Mass spectrometry analysis revealed that the purified complex produces cellulose chains with a degree of polymerization of at least 80 residues. Truncation analyses demonstrated that the removal of a significant extracellular segment of GlxA had no impact on complex formation, but significantly diminished activity of CslA. Altogether, our work demonstrates that CslA and GlxA form the active core of the cellulose synthase complex and provide molecular insights into a unique cellulose biosynthesis system that is conserved in streptomycetes. IMPORTANCE: Cellulose stands out as the most abundant polysaccharide on Earth. While the synthesis of this polysaccharide has been extensively studied in plants and Gram-negative bacteria, the mechanisms in Gram-positive bacteria have remained largely unknown. Our research unveils a novel cellulose synthase complex formed by the interaction between the cellulose synthase-like protein CslA and the radical copper oxidase GlxA from Streptomyces lividans, a soil-dwelling Gram-positive bacterium. This discovery provides molecular insights into the distinctive cellulose biosynthesis machinery. Beyond expanding our understanding of cellulose biosynthesis, this study also opens avenues for exploring biotechnological applications and ecological roles of cellulose in Gram-positive bacteria, thereby contributing to the broader field of microbial cellulose biosynthesis and biofilm research.

Apolipoprotein-CIII O-Glycosylation Is Associated with Micro- and Macrovascular Complications of Type 2 Diabetes.

Apolipoprotein-CIII (apo-CIII) inhibits the clearance of triglycerides from circulation and is associated with an increased risk of diabetes complications. It exists in four main proteoforms: O-glycosylated variants containing either zero, one, or two sialic acids and a non-glycosylated variant. O-glycosylation may affect the metabolic functions of apo-CIII. We investigated the associations of apo-CIII glycosylation in blood plasma, measured by mass spectrometry of the intact protein, and genetic variants with micro- and macrovascular complications (retinopathy, nephropathy, neuropathy, cardiovascular disease) of type 2 diabetes in a DiaGene study (n = 1571) and the Hoorn DCS cohort (n = 5409). Mono-sialylated apolipoprotein-CIII (apo-CIII1) was associated with a reduced risk of retinopathy (ß = -7.215, 95% CI -11.137 to -3.294) whereas disialylated apolipoprotein-CIII (apo-CIII2) was associated with an increased risk (ß = 5.309, 95% CI 2.279 to 8.339). A variant of the GALNT2-gene (rs4846913), previously linked to lower apo-CIII0a, was associated with a decreased prevalence of retinopathy (OR = 0.739, 95% CI 0.575 to 0.951). Higher apo-CIII1 levels were associated with neuropathy (ß = 7.706, 95% CI 2.317 to 13.095) and lower apo-CIII0a with macrovascular complications (ß = -9.195, 95% CI -15.847 to -2.543). In conclusion, apo-CIII glycosylation was associated with the prevalence of micro- and macrovascular complications of diabetes. Moreover, a variant in the GALNT2-gene was associated with apo-CIII glycosylation and retinopathy, suggesting a causal effect. The findings facilitate a molecular understanding of the pathophysiology of diabetes complications and warrant consideration of apo-CIII glycosylation as a potential target in the prevention of diabetes complications.

Revealing charge heterogeneity of stressed trastuzumab at the subunit level.

Trastuzumab is known to be heterogeneous in terms of charge. Stressing trastuzumab under physiological conditions (pH 7.4 and 37 °C) increases charge heterogeneity further. Separation of charge variants of stressed trastuzumab at the intact protein level is challenging due to increasing complexity making it difficult to obtain pure charge variants for further characterization. Here we report an approach for revealing charge heterogeneity of stressed trastuzumab at the subunit level by pH gradient cation-exchange chromatography. Trastuzumab subunits were generated after limited proteolytic cleavage with papain, IdeS, and GingisKHAN®. The basic pI of Fab and F(ab)2 fragments allowed to use the same pH gradient for intact protein and subunit level analysis. Baseline separation of Fab subunits was obtained after GingisKHAN® and papain digestion and the corresponding modifications were determined by LC-MS/MS peptide mapping and middle-down MALDI-ISD FT-ICR MS. The described approach allows a comprehensive charge variant analysis of therapeutic antibodies that have two or more modification sites in the Fab region.

Apolipoprotein-CIII O-Glycosylation, a Link between GALNT2 and Plasma Lipids.

Apolipoprotein-CIII (apo-CIII) is involved in triglyceride-rich lipoprotein metabolism and linked to beta-cell damage, insulin resistance, and cardiovascular disease. Apo-CIII exists in four main proteoforms: non-glycosylated (apo-CIII0a), and glycosylated apo-CIII with zero, one, or two sialic acids (apo-CIII0c, apo-CIII1 and apo-CIII2). Our objective is to determine how apo-CIII glycosylation affects lipid traits and type 2 diabetes prevalence, and to investigate the genetic basis of these relations with a genome-wide association study (GWAS) on apo-CIII glycosylation. We conducted GWAS on the four apo-CIII proteoforms in the DiaGene study in people with and without type 2 diabetes (n = 2318). We investigated the relations of the identified genetic loci and apo-CIII glycosylation with lipids and type 2 diabetes. The associations of the genetic variants with lipids were replicated in the Diabetes Care System (n = 5409). Rs4846913-A, in the GALNT2-gene, was associated with decreased apo-CIII0a. This variant was associated with increased high-density lipoprotein cholesterol and decreased triglycerides, while high apo-CIII0a was associated with raised high-density lipoprotein-cholesterol and triglycerides. Rs67086575-G, located in the IFT172-gene, was associated with decreased apo-CIII2 and with hypertriglyceridemia. In line, apo-CIII2 was associated with low triglycerides. On a genome-wide scale, we confirmed that the GALNT2-gene plays a major role i O-glycosylation of apolipoprotein-CIII, with subsequent associations with lipid parameters. We newly identified the IFT172/NRBP1 region, in the literature previously associated with hypertriglyceridemia, as involved in apolipoprotein-CIII sialylation and hypertriglyceridemia. These results link genomics, glycosylation, and lipid metabolism, and represent a key step towards unravelling the importance of O-glycosylation in health and disease.

Glycan and Protein Analysis of Glycoengineered Bacterial E. coli Vaccines by MALDI-in-Source Decay FT-ICR Mass Spectrometry.

Bacterial glycoconjugate vaccines have a major role in preventing microbial infections. Immunogenic bacterial glycans, such as O-antigen polysaccharides, can be recombinantly expressed and combined with specific carrier proteins to produce effective vaccines. O-Antigen polysaccharides are typically polydisperse, and carrier proteins can have multiple glycosylation sites. Consequently, recombinant glycoconjugate vaccines have a high structural heterogeneity, making their characterization challenging. Since development and quality control processes rely on such characterization, novel strategies are needed for faster and informative analysis. Here, we present a novel approach employing minimal sample preparation and ultrahigh-resolution mass spectrometry analysis for protein terminal sequencing and characterization of the oligosaccharide repeat units of bacterial glycoconjugate vaccines. Three glycoconjugate vaccine candidates, obtained from the bioconjugation of the O-antigen polysaccharides from E. coli serotypes O2, O6A, and O25B with the genetically detoxified exotoxin A from Pseudomonas aeruginosa, were analyzed by MALDI-in-source decay (ISD) FT-ICR MS. Protein and glycan ISD fragment ions were selectively detected using 1,5-diaminonaphtalene and a 2,5-dihydroxybenzoic acid/2-hydroxy-5-methoxybenzoic acid mixture (super-DHB) as a MALDI matrix, respectively. The analysis of protein fragments required the absence of salts in the samples, while the presence of salt was key for the detection of sodiated glycan fragments. MS/MS analysis of O-antigen ISD fragments allowed for the detection of specific repeat unit signatures. The developed strategy requires minute sample amounts, avoids the use of chemical derivatizations, and comes with minimal hands-on time allowing for fast corroboration of key structural features of bacterial glycoconjugate vaccines during early- and late-stage development.

Native Liquid Chromatography and Mass Spectrometry to Structurally and Functionally Characterize Endo-Xylanase Proteoforms.

Xylanases are of great value in various industries, including paper, food, and biorefinery. Due to their biotechnological production, these enzymes can contain a variety of post-translational modifications, which may have a profound effect on protein function. Understanding the structure-function relationship can guide the development of products with optimal performance. We have developed a workflow for the structural and functional characterization of an endo-1,4-ß-xylanase (ENDO-I) produced by Aspergillus niger with and without applying thermal stress. This workflow relies on orthogonal native separation techniques to resolve proteoforms. Mass spectrometry and activity assays of separated proteoforms permitted the establishment of structure-function relationships. The separation conditions were focus on balancing efficient separation and protein functionality. We employed size exclusion chromatography (SEC) to separate ENDO-I from other co-expressed proteins. Charge variants were investigated with ion exchange chromatography (IEX) and revealed the presence of low abundant glycated variants in the temperature-stressed material. To obtain better insights into the effect on glycation on function, we enriched for these species using boronate affinity chromatography (BAC). The activity measurements showed lower activity of glycated species compared to the non-modified enzyme. Altogether, this workflow allowed in-depth structural and functional characterization of ENDO-I proteoforms.

Analysis of Synthetic Monodisperse Polysaccharides by Wide Mass Range Ultrahigh-Resolution MALDI Mass Spectrometry.

Carbohydrates, such as oligo- and polysaccharides, are highly abundant biopolymers that are involved in numerous processes. The study of their structure and functions is commonly based on a material that is isolated from complex natural sources. However, a more precise analysis requires pure compounds with well-defined structures that can be obtained from chemical or enzymatic syntheses. Novel synthetic strategies have increased the accessibility of larger monodisperse polysaccharides, posing a challenge to the analytical methods used for their molecular characterization. Here, we present wide mass range ultrahigh-resolution matrix-assisted laser desorption/ionization (MALDI) Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) as a powerful platform for the analysis of synthetic oligo- and polysaccharides. Synthetic carbohydrates 16-, 64-, 100-, and 151-mers were mass analyzed and characterized by MALDI in-source decay FT-ICR MS. Detection of fragment ions generated from glycosidic bond cleavage (or cross-ring cleavage) provided information of the monosaccharide content and the linkage type, allowing for the corroboration of the carbohydrate compositions and structures.

A semi-automated, high throughput approach for O-glycosylation profiling of in vitro established cancer cell lines by MALDI-FT-ICR MS.

The study of protein O-glycosylation is important in biological research as O-glycans have been reported to regulate a multitude of molecular and cell biology processes occurring in cancer. It is known that alterations in O-glycosylation are involved in the development and progression of cancer. Their easy accessibility makes in vitro established cell lines suitable and useful models for studying biological mechanisms in disease. However, the O-glycosylation analysis of large numbers of samples, as required in systems biology and biomarker discovery studies, is often challenging. In the present study, O-glycans from three human colorectal cancer cell lines and two human pancreatic cancer cell lines were released by semi-automated, high throughput reductive ß-elimination and analysed using ultrahigh resolution MALDI-FT-ICR MS. Automated data integration and processing was performed using MassyTools, where the analyte was automatically included for relative quantitation based on a range of selection criteria including signal-to-noise ratio, mass error and isotopic pattern quality scores. A total of 126 O-glycan compositions, ranging from a single monosaccharide to large oligosaccharides exhibiting complex glycan motifs, were detected. The use of ultrahigh resolution MALDI-FTICR MS enabled glycan identification and quantitation in the matrix region of the spectrum. This approach has the potential to be used for O-glycosylation analysis of large numbers of samples, such as patient sample cohorts.

Serum N-glycan profiles differ for various breast cancer subtypes.

Breast cancer is the most prevalent cancer in women. Early detection of this disease improves survival and therefore population screenings, based on mammography, are performed. However, the sensitivity of this screening modality is not optimal and new screening methods, such as blood tests, are being explored. Most of the analyses that aim for early detection focus on proteins in the bloodstream. In this study, the biomarker potential of total serum N-glycosylation analysis was explored with regard to detection of breast cancer. In an age-matched case-control setup serum protein N-glycan profiles from 145 breast cancer patients were compared to those from 171 healthy individuals. N-glycans were enzymatically released, chemically derivatized to preserve linkage-specificity of sialic acids and characterized by high resolution mass spectrometry. Logistic regression analysis was used to evaluate associations of specific N-glycan structures as well as N-glycosylation traits with breast cancer. In a case-control comparison three associations were found, namely a lower level of a two triantennary glycans and a higher level of one tetraantennary glycan in cancer patients. Of note, various other N-glycomic signatures that had previously been reported were not replicated in the current cohort. It was further evaluated whether the lack of replication of breast cancer N-glycomic signatures could be partly explained by the heterogenous character of the disease since the studies performed so far were based on cohorts that included diverging subtypes in different numbers. It was found that serum N-glycan profiles differed for the various cancer subtypes that were analyzed in this study.

Improved N- and C-Terminal Sequencing of Proteins by Combining Positive and Negative Ion MALDI In-Source Decay Mass Spectrometry.

The development of various ionization and fragmentation techniques has been of key importance for establishing mass spectrometry (MS) as a powerful tool for protein characterization. One example of this is matrix-assisted laser desorption/ionization (MALDI) combined with in-source decay (ISD) fragmentation that allows mapping of N- and C-terminal regions of large proteins without the need for proteolysis. Positive ion mode ISD fragments are commonly assigned in the mass region above m/z 1000, while MALDI matrix ions generally hamper the detection of smaller singly charged fragments. The ultrahigh resolving power provided by Fourier transform ion cyclotron resonance (FT-ICR) MS partially overcomes this limitation, but to further increase the detection of smaller fragments we have revisited the application of negative ion mode MALDI-ISD and found good coverage of the peptide chain termini starting from c'2 and z'2 fragment ions. For the first time, we demonstrate that the combination of negative and positive ion MALDI FT-ICR MS is a useful tool to improve the characterization of mAbs. The different specificities of the two ion modes allowed us to selectively cover the sequence of the light and heavy chains of mAbs at increased sensitivity. A comprehensive evaluation of positive and negative ion mode MALDI-ISD FT-ICR MS in the m/z range 46-13 500 showed an increased sequence coverage for three standard proteins, namely, myoglobin, SiLuLite mAb, and NIST mAb. The data obtained in the two ion modes were, in part, complementary.

Evaluation of Sibling and Twin Fragment Ions Improves the Structural Characterization of Proteins by Top-Down MALDI In-Source Decay Mass Spectrometry.

Comprehensive determination of primary sequence and identification of post-translational modifications (PTMs) are key elements in protein structural analysis. Various mass spectrometry (MS) based fragmentation techniques are powerful approaches for mapping both the amino acid sequence and PTMs; one of these techniques is matrix-assisted laser desorption/ionization (MALDI), combined with in-source decay (ISD) fragmentation and Fourier-transform ion cyclotron resonance (FT-ICR) MS. MALDI-ISD MS protein analysis involves only minimal sample preparation and does not require spectral deconvolution. The resulting MALDI-ISD MS data is complementary to electrospray ionization-based MS/MS sequencing readouts, providing knowledge on the types of fragment ions is available. In this study, we evaluate the isotopic distributions of z' ions in protein top-down MALDI-ISD FT-ICR mass spectra and show why these distributions can deviate from theoretical profiles as a result of co-occurring and isomeric z and y-NH3 ions. Two synthetic peptides, containing either normal or deuterated alanine residues, were used to confirm the presence and unravel the identity of isomeric z and y-NH3 fragment ions ("twins"). Furthermore, two reducing MALDI matrices, namely 1,5-diaminonaphthalene and N-phenyl-p-phenylenediamine were applied that yield ISD mass spectra with different fragment ion distributions. This study demonstrates that the relative abundance of isomeric z and y-NH3 ions requires consideration for accurate and confident assignments of z' ions in MALDI-ISD FT-ICR mass spectra.

Structural Analysis of Monoclonal Antibodies by Ultrahigh Resolution MALDI In-Source Decay FT-ICR Mass Spectrometry.

The emergence of complex protein therapeutics in general and monoclonal antibodies (mAbs) in particular have stimulated analytical chemists to develop new methods and strategies for their structural characterization. Mass spectrometry plays a key role in providing information on the primary amino acid sequence, post-translational modifications, and other structure characteristics that must be monitored during the manufacturing process and subsequent quality control assessment. In this study, we present a novel method that allows structural characterization of mAbs based on MALDI in-source decay (ISD) fragmentation, coupled with Fourier transform ion cyclotron resonance (FT-ICR) MS. The method benefits from higher resolution of absorption mode FT mass spectra, compared to magnitude mode, which enables simultaneous identification of ISD fragments from both the heavy and light chains with a higher confidence in a wide mass range up to m/ z 13 500. This method was applied to two standard mAbs, namely NIST mAb and trastuzumab, in preparation for method application in an interlaboratory study on mAbs structural analysis coordinated by the Consortium for Top-Down Proteomics. Extensive sequence coverage was obtained from the middle-down analysis (IdeS- and GingisKHAN-digested mAbs) that complemented the top-down analysis of intact mAbs. In addition, MALDI FT-ICR MS of IdeS-digested mAbs allowed isotopic-level profiling of proteoforms with regard to heavy chain N-glycosylation.

Automated Plasma Glycomics with Linkage-Specific Sialic Acid Esterification and Ultrahigh Resolution MS.

High-throughput mass spectrometry (MS) glycomics is an emerging field driven by technological advancements including sample preparation and data processing. Previously, we reported an automated protocol for the analysis of N-glycans released from plasma proteins that included sialic acid derivatization with linkage-specificity, namely, ethylation of α2,6-linked sialic acid residues and lactone formation of α2,3-linked sialic acids. In the current study, each step in this protocol was further optimized. Method improvements included minimizing the extent of side-reaction during derivatization, an adjusted glycan purification strategy and mass analysis of the released N-glycans by ultrahigh resolution matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance MS. The latter resolved peak overlap and simplified spectral alignment due to high mass measurement precision. Moreover, this resulted in more confident glycan assignments and improved signal-to-noise for low-abundant species. The performance of the protocol renders high-throughput applications feasible in the exciting field of clinical glycomics.

Accounting for isotopic clustering in Fourier transform mass spectrometry data analysis for clinical diagnostic studies.

Mass spectrometry based clinical proteomics has emerged as a powerful tool for high-throughput protein profiling and biomarker discovery. Recent improvements in mass spectrometry technology have boosted the potential of proteomic studies in biomedical research. However, the complexity of the proteomic expression introduces new statistical challenges in summarizing and analyzing the acquired data. Statistical methods for optimally processing proteomic data are currently a growing field of research. In this paper we present simple, yet appropriate methods to preprocess, summarize and analyze high-throughput MALDI-FTICR mass spectrometry data, collected in a case-control fashion, while dealing with the statistical challenges that accompany such data. The known statistical properties of the isotopic distribution of the peptide molecules are used to preprocess the spectra and translate the proteomic expression into a condensed data set. Information on either the intensity level or the shape of the identified isotopic clusters is used to derive summary measures on which diagnostic rules for disease status allocation will be based. Results indicate that both the shape of the identified isotopic clusters and the overall intensity level carry information on the class outcome and can be used to predict the presence or absence of the disease.

Typing Pseudomonas aeruginosa Isolates with Ultrahigh Resolution MALDI-FTICR Mass Spectrometry.

The introduction of standardized matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) platforms in the medical microbiological practice has revolutionized the way microbial species identification is performed on a daily basis. To a large extent, this is due to the ease of operation. Acquired spectra are compared to profiles obtained from cultured colonies present in a reference spectra database. It is fast and reliable, and costs are low compared to previous diagnostic approaches. However, the low resolution and dynamic range of the MALDI-TOF profiles have shown limited applicability for the discrimination of different bacterial strains, as achieved with typing based on genetic markers. This is pivotal in cases where certain strains are associated with, e.g., virulence or antibiotic resistance. Ultrahigh resolution MALDI-FTICR MS allows the measurement of small proteins at isotopic resolution and can be used to analyze complex mixtures with increased dynamic range and higher precision than MALDI-TOF MS, while still generating results in a similar time frame. Here, we propose to use ultrahigh resolution 15T MALDI-Fourier transform ion cyclotron resonance (FTICR) MS to discriminate clinically relevant bacterial strains after species identification performed by MALDI-TOF MS. We used a collection of well characterized Pseudomonas aeruginosa strains, featuring distinct antibiotic resistance profiles, and isolates obtained during hospital outbreaks. Following cluster analysis based on amplification fragment length polymorphism (AFLP), these strains were grouped into three different clusters. The same clusters were obtained using protein profiles generated by MALDI-FTICR MS. Subsequent intact protein analysis by electrospray ionization (ESI)-collision-induced dissociation (CID)-FTICR MS was applied to identify protein isoforms that contribute to the separation of the different clusters, illustrating the additional advantage of this analytical platform.

Top-down MALDI-in-source decay-FTICR mass spectrometry of isotopically resolved proteins.

An accurate mass measurement of a known protein provides information on potential amino acid deletions and post-translational modifications. Although this field is dominated by strategies based on electrospray ionization, mass spectrometry (MS) methods using matrix-assisted laser desorption/ionization (MALDI) have the advantage of yielding predominantly singly charged precursor ions, thus avoiding peak overlap from different charge states of multiple species. Such MALDI-MS methods require mass measurement at ultrahigh resolution, which is provided by Fourier transform ion cyclotron resonance (FTICR) mass analyzers. Recently, using a MALDI-FTICR-MS platform equipped with a 15 T magnet, we reported on the mass analysis of intact human serum peptides and small proteins with isotopic resolution up to ∼15 kDa and identified new proteoforms from an accurate measurement of mass distances. In the current study, we have used this FTICR system after an upgrade with a novel dynamically harmonized ICR cell, i.e., ParaCell, for mapping isotopically resolved intact proteins up to about 17 kDa and performed top-down MALDI in-source decay (ISD) analysis. Standard proteins myoglobin (m/z-value 16,950) and ribonuclease B (m/z-value 14,900) were measured with resolving powers of 62,000 and 61,000, respectively. Furthermore, it will be shown that (singly charged) MALDI-ISD fragment ions can be measured at isotopic resolution up to m/z-value 12,000 (e.g., resolving power 39,000 at m/z-value 12,000) providing more reliable identifications. Moreover, examples are presented of pseudo-MS(3) experiments on ISD fragment ions from RNase B by collisional-induced dissociation (CID).

Structure characterization of unexpected covalent O-sulfonation and ion-pairing on an extremely hydrophilic peptide with CE-MS and FT-ICR-MS.

In this study, we characterized unexpected side-products in a commercially synthesized peptide with the sequence RPRTRLHTHRNR. This so-called peptide D3 was selected by mirror phage display against low molecular weight amyloid-ß-peptide (Aß) associated with Alzheimer's disease. Capillary electrophoresis (CE) was the method of choice for structure analysis because the extreme hydrophilicity of the peptide did not allow reversed-phase liquid chromatography (RPLC) and hydrophilic interaction stationary phases (HILIC). CE-MS analysis, applying a strongly acidic background electrolyte and different statically adsorbed capillary coatings, provided fast and efficient analysis and revealed that D3 unexpectedly showed strong ion-pairing with sulfuric acid. Moreover, covalent O-sulfonation at one or two threonine residues was identified as a result of a side reaction during peptide synthesis, and deamidation was found at either the asparagine residue or at the C-terminus. In total, more than 10 different species with different m/z values were observed. Tandem-MS analysis with collision induced dissociation (CID) using a CE-quadrupole-time-of-flight (QTOF) setup predominantly resulted in sulfate losses and did not yield any further characteristic fragment ions at high collision energies. Therefore, direct infusion Fourier transform ion cyclotron resonance (FT-ICR) MS was employed to identify the covalent modification and discriminate O-sulfonation from possible O-phosphorylation by using an accurate mass analysis. Electron transfer dissociation (ETD) was used for the identification of the threonine O-sulfation sites. In this work, it is shown that the combination of CE-MS and FT-ICR-MS with ETD fragmentation was essential for the full characterization of this extremely basic peptide with labile modifications.

Developments in FTICR-MS and Its Potential for Body Fluid Signatures.

Fourier transform mass spectrometry (FTMS) is the method of choice for measurements that require ultra-high resolution. The establishment of Fourier transform ion cyclotron resonance (FTICR) MS, the availability of biomolecular ionization techniques and the introduction of the Orbitrap™ mass spectrometer have widened the number of FTMS-applications enormously. One recent example involves clinical proteomics using FTICR-MS to discover and validate protein biomarker signatures in body fluids such as serum or plasma. These biological samples are highly complex in terms of the type and number of components, their concentration range, and the structural identity of each species, and thus require extensive sample cleanup and chromatographic separation procedures. Clearly, such an elaborate and multi-step sample preparation process hampers high-throughput analysis of large clinical cohorts. A final MS read-out at ultra-high resolution enables the analysis of a more complex sample and can thus simplify upfront fractionations. To this end, FTICR-MS offers superior ultra-high resolving power with accurate and precise mass-to-charge ratio (m/z) measurement of a high number of peptides and small proteins (up to 20 kDa) at isotopic resolution over a wide mass range, and furthermore includes a wide variety of fragmentation strategies to characterize protein sequence and structure, including post-translational modifications (PTMs). In our laboratory, we have successfully applied FTICR "next-generation" peptide profiles with the purpose of cancer disease classifications. Here we will review a number of developments and innovations in FTICR-MS that have resulted in robust and routine procedures aiming for ultra-high resolution signatures of clinical samples, exemplified with state-of-the-art examples for serum and saliva.

Structural analysis of an intact monoclonal antibody by online electrochemical reduction of disulfide bonds and Fourier transform ion cyclotron resonance mass spectrometry.

Structural confirmation and quality control of recombinant monoclonal antibodies (mAbs) by top-down mass spectrometry is still challenging due to the size of the proteins, disulfide content, and post-translational modifications such as glycosylation. In this study we have applied electrochemistry (EC) to overcome disulfide bridge complexity in top-down analysis of mAbs. To this end, an electrochemical cell was coupled directly to an electrospray ionization (ESI) source and a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer (MS) equipped with a 15 T magnet. By performing online EC-assisted reduction of interchain disulfide bonds in an intact mAb, the released light chains could be selected for tandem mass spectrometry (MS/MS) analysis without interference from heavy-chain fragments. Moreover, the acquisition of full MS scans under denaturing conditions allowed profiling of all abundant mAb glycoforms. Ultrahigh-resolution FTICR-MS measurements provided fully resolved isotopic distributions of intact mAb and enabled the identification of the most abundant adducts and other interfering species. Furthermore, it was found that reduction of interchain disulfide bonds occurs in the ESI source dependent on capillary voltage and solvent composition. This phenomenon was systematically evaluated and compared with the results obtained from reduction in the electrochemical cell.