Can We Boost N-Glycopeptide Identification Confidence? Smart Collision Energy Choice Taking into Account Structure and Search Engine.

High confidence and reproducibility are still challenges in bottom-up mass spectrometric N-glycopeptide identification. The collision energy used in the MS/MS measurements and the database search engine used to identify the species are perhaps the two most decisive factors. We investigated how the structural features of N-glycopeptides and the choice of the search engine influence the optimal collision energy, delivering the highest identification confidence. We carried out LC-MS/MS measurements using a series of collision energies on a large set of N-glycopeptides with both the glycan and peptide part varied and studied the behavior of Byonic, pGlyco, and GlycoQuest scores. We found that search engines show a range of behavior between peptide-centric and glycan-centric, which manifests itself already in the dependence of optimal collision energy on m/z. Using classical statistical and machine learning methods, we revealed that peptide hydrophobicity, glycan and peptide masses, and the number of mobile protons also have significant and search-engine-dependent influence, as opposed to a series of other parameters we probed. We envisioned an MS/MS workflow making a smart collision energy choice based on online available features such as the hydrophobicity (described by retention time) and glycan mass (potentially available from a scout MS/MS). Our assessment suggests that this workflow can lead to a significant gain (up to 100%) in the identification confidence, particularly for low-scoring hits close to the filtering limit, which has the potential to enhance reproducibility of N-glycopeptide analyses. Data are available via MassIVE (MSV000093110).

Collision energies: Optimization strategies for bottom-up proteomics.

Mass-spectrometry coupled to liquid chromatography is an indispensable tool in the field of proteomics. In the last decades, more and more complex and diverse biochemical and biomedical questions have arisen. Problems to be solved involve protein identification, quantitative analysis, screening of low abundance modifications, handling matrix effect, and concentrations differing by orders of magnitude. This led the development of more tailored protocols and problem centered proteomics workflows, including advanced choice of experimental parameters. In the most widespread bottom-up approach, the choice of collision energy in tandem mass spectrometric experiments has outstanding role. This review presents the collision energy optimization strategies in the field of proteomics which can help fully exploit the potential of MS based proteomics techniques. A systematic collection of use case studies is then presented to serve as a starting point for related further scientific work. Finally, this article discusses the issue of comparing results from different studies or obtained on different instruments, and it gives some hints on methodology transfer between laboratories based on measurement of reference species.

Very Low-Pressure CID Experiments: High Energy Transfer and Fragmentation Pattern at the Single Collision Regime.

We have performed CID experiments on a triple quadrupole instrument, lowering the collision gas pressure by 50 times compared to its conventional value. The results show that at very low-collision gas pressure, single collisions dominate the spectra. Indirectly, these results suggest that under conventional conditions, 20-50 collisions may be typical in CID experiments. The results show a marked difference between low- and high-pressure CID spectra, the latter being characterized in terms of 'slow heating' and predominance of consecutive reactions. The results indicate that under single collision conditions, the collisional energy transfer efficiency is very high: nearly 100% of the center of mass kinetic energy is converted to internal energy.

Diversity Matters: Optimal Collision Energies for Tandem Mass Spectrometric Analysis of a Large Set of N-Glycopeptides.

Identification and characterization of N-glycopeptides from complex samples are usually based on tandem mass spectrometric measurements. Experimental settings, especially the collision energy selection method, fundamentally influence the obtained fragmentation pattern and hence the confidence of the database search results ("score"). Using standards of naturally occurring glycoproteins, we mapped the Byonic and pGlyco search engine scores of almost 200 individual N-glycopeptides as a function of collision energy settings on a quadrupole time of flight instrument. The resulting unprecedented amount of peptide-level information on such a large and diverse set of N-glycopeptides revealed that the peptide sequence heavily influences the energy for the highest score on top of an expected general linear trend with m/z. Search engine dependence may also be noteworthy. Based on the trends, we designed an experimental method and tested it on HeLa, blood plasma, and monoclonal antibody samples. As compared to the literature, these notably lower collision energies in our workflow led to 10-50% more identified N-glycopeptides, with higher scores. We recommend a simple approach based on a small set of reference N-glycopeptides easily accessible from glycoprotein standards to ease the precise determination of optimal methods on other instruments. Data sets can be accessed via the MassIVE repository (MSV000089657 and MSV000090218).

The Role of IgG Fc Region N-Glycosylation in the Pathomechanism of Rheumatoid Arthritis.

Anti-citrullinated protein antibodies (ACPAs) are involved in the pathogenesis of rheumatoid arthritis. N-glycosylation pattern of ACPA-IgG and healthy IgG Fc differs. The aim of this study is to determine the relative sialylation and galactosylation level of ACPAs and control IgG to assess their capability of inducing TNFα production, and furthermore, to analyze the correlations between the composition of Fc glycans and inflammatory markers in RA. We isolated IgG from sera of healthy volunteers and RA patients, and purified ACPAs on a citrulline-peptide column. Immunocomplexes (IC) were formed by adding an F(ab)2 fragment of anti-human IgG. U937 cells were used to monitor the binding of IC to FcγR and to trigger TNFα release determined by ELISA. To analyze glycan profiles, control IgG and ACPA-IgG were digested with trypsin and the glycosylation patterns of glycopeptides were analyzed by determining site-specific N-glycosylation using nano-UHPLC-MS/MS. We found that both sialylation and galactosylation levels of ACPA-IgG negatively correlate with inflammation-related parameters such as CRP, ESR, and RF. Functional assays show that dimerized ACPA-IgG significantly enhances TNFα release in an FcγRI-dependent manner, whereas healthy IgG does not. TNFα production inversely correlates with the relative intensities of the G0 glycoform, which lacks galactose and terminal sialic acid moieties.

Alterations in protein expression and site-specific N-glycosylation of prostate cancer tissues.

Identifying molecular alterations occurring during cancer progression is essential for a deeper understanding of the underlying biological processes. Here we have analyzed cancerous and healthy prostate biopsies using nanoLC-MS(MS) to detect proteins with altered expression and N-glycosylation. We have identified 75 proteins with significantly changing expression during disease progression. The biological processes involved were assigned based on protein-protein interaction networks. These include cellular component organization, metabolic and localization processes. Multiple glycoproteins were identified with aberrant glycosylation in prostate cancer, where differences in glycosite-specific sialylation, fucosylation, and galactosylation were the most substantial. Many of the glycoproteins with altered N-glycosylation were extracellular matrix constituents, and are heavily involved in the establishment of the tumor microenvironment.

Influence of Post-Translational Modifications on Protein Identification in Database Searches.

Comprehensive analysis of post-translation modifications (PTMs) is an important mission of proteomics. However, the consideration of PTMs increases the search space and may therefore impair the efficiency of protein identification. Using thousands of proteomic searches, we investigated the practical aspects of considering multiple PTMs in Byonic searches for the maximization of protein and peptide hits. The inclusion of all PTMs, which occur with at least 2% frequency in the sample, has an advantageous effect on protein and peptide identification. A linear relationship was established between the number of considered PTMs and the number of reliably identified peptides and proteins. Even though they handle multiple modifications less efficiently, the results of MASCOT (using the Percolator function) and Andromeda (the search engine included in MaxQuant) became comparable to those of Byonic, in the case of a few PTMs.

Stability and recovery issues concerning chondroitin sulfate disaccharide analysis.

Chondroitin sulfate (CS) is a widely studied class of glycosaminoglycans, responsible for diverse biological functions. Structural analysis of CS is generally based on disaccharide analysis. Sample preparation is a key analytical issue in this case. However, a detailed study on the stability and recovery of CS-derived species has been lacking so far. We have found that for solvent exchange, in general, vacuum evaporation (SpeedVac) is much preferable than lyophilization. Moreover, in the case of aqueous solutions, higher recovery was experienced than in solutions with high organic solvent content. Storage of the resulting disaccharide mixture in typical HPLC injection solvents is also critical; decomposition starts after 12 h at 4 °C; therefore, the mixtures should not be kept in the sample tray of an automatic injector for a long time. The study, therefore, lays down suggestions on proper sample preparation and measurement conditions for biologically derived chondroitin sulfate species.

Collision energies on QTof and Orbitrap instruments: How to make proteomics measurements comparable?

Quadrupole time-of-flight (QTof) collision-induced dissociation (CID) and Orbitrap higher-energy collisional dissociation (HCD) are the most commonly used fragmentation techniques in mass spectrometry-based proteomics workflows. The information content of the MS/MS spectra is first and foremost determined by the applied collision energy. How can we set up the two instrument types to achieve maximum transferability? To answer this question, we compared MS/MS spectra obtained on a Bruker QTof CID and a Thermo Q-Exactive Focus Orbitrap HCD instrument as a function of collision energy using the similarity index. Results show that with a few eV lower collision energy setting on HCD (Orbitrap-specific CID) than on QTof CID, nearly identical MS/MS spectra can be obtained for leucine enkephalin pentapeptide standard, for selected +2 and +3 enolase tryptic peptides and for a large number of peptides in a HeLa protein digest. The Bruker QTof was able to produce colder ions, which may be significant to study inherently labile compounds. Further, we examined energy dependence of peptide identification confidence, as characterized by Mascot scores, on the HeLa peptides. In line with earlier QTof results, this dependence shows one or two maxima (unimodal or bimodal behavior) on Orbitrap. The fraction of bimodal peptides is lower on Orbitrap. Optimal energies as a function of m/z show a similar linear trend on both instruments, which suggests that with appropriate collision energy adjustment, matching conditions for proteomics can be achieved. Data have been deposited in the MassIVE repository (MSV000086434).

Tailoring to Search Engines: Bottom-Up Proteomics with Collision Energies Optimized for Identification Confidence.

Bottom-up proteomics relies on identification of peptides from tandem mass spectra, usually via matching against sequence databases. Confidence in a peptide-spectrum match can be characterized by a score value given by the database search engines, and it depends on the information content and the quality of the spectrum. The latter are influenced by experimental parameters, of which the collision energy is the most important one in the case of collision-induced dissociation. We examined how the identification score of the Byonic and Andromeda (MaxQuant) engines varies with collision energy for more than a thousand individual peptides from a HeLa tryptic digest on a QTof instrument. We thereby extended our earlier study on Mascot scores and corroborated its findings on the potential bimodal nature of this energy dependence. Optimal energies as a function of m/z show comparable linear trends for the three engines. On the basis of peptide-level results, we designed methods with one or two liquid chromatography-tandem mass spectrometry (LC-MS/MS) runs and various collision energy settings and assessed their practical performance in peptide and protein identification from the HeLa standard sample. A 10-40% gain in various measures, such as the number of identified proteins or sequence coverage, was obtained over the factory default settings. Best performing methods differ for the three engines, suggesting that the experimental parameters should be fine-tuned to the choice of the engine. We also recommend a simple approach and provide reference data to ease the transfer of the optimized methods to other mass spectrometers relevant for proteomics. We demonstrate the utility of this approach on an Orbitrap instrument. Data sets can be accessed via the MassIVE repository (MSV000086379).

Salt gradient chromatographic separation of chondroitin sulfate disaccharides.

In the present study, we describe the development of a fast, 2-step salt gradient for analysis of chondroitin sulfate disaccharides. Using salt gradients, which is somewhat unusual in HILIC-based separations, provides relatively fast chromatography with excellent sensitivity (15 min cycle time, 10-20 fmol/µL detection, 30-50 fmol/µL quantitation limit), and good linearity. The efficiency of the new method is demonstrated by measuring human tissue slices of healthy, cirrhotic, and cancerous liver samples. Preliminary results show major differences among the quantity and sulfation pattern of the various sample types.

Changes in the proteome of sea urchin Paracentrotus lividus coelomocytes in response to LPS injection into the body cavity.

BACKGROUND: The immune system of echinoderm sea urchins is characterised by a high degree of complexity that is not completely understood. The Mediterranean sea urchin Paracentrotus lividus coelomocytes mediate immune responses through phagocytosis, encapsulation of non-self particles, and production of diffusible factors including antimicrobial molecules. Details of these processes, and molecular pathways driving these mechanisms, are still to be fully elucidated. PRINCIPAL FINDINGS: In the present study we treated the sea urchin P. lividus with the bacterial lipopolysaccharide (LPS) and collected coelomocytes at different time-points (1, 3, 6 and 24 hours). We have shown, using label-free quantitative mass spectrometry, how LPS is able to modulate the coelomocyte proteome and to effect cellular pathways, such as endocytosis and phagocytosis, as soon as the immunomodulating agent is injected. The present study has also shown that treatment can modulate various cellular processes such as cytoskeleton reorganisation, and stress and energetic homeostasis. CONCLUSIONS: Our data demonstrates, through mass spectrometry and the following functional annotation bioinformatics analysis, how the bacterial wall constituent is sufficient to set off an immune response inducing cytoskeleton reorganisation, the appearance of clusters of heat shock proteins (Hsp) and histone proteins and the activation of the endocytic and phagocytic pathways. Data are available via ProteomeXchange with identifier PXD008439.

Salt and solvent effects in the microscale chromatographic separation of heparan sulfate disaccharides.

The analysis of heparan sulfate disaccharides poses a real challenge both from chromatographic and mass spectrometric point of view. This necessitates the constant improvement of their analytical methodology. In the present study, the chromatographic effects of solvent composition, salt concentration, and salt type were systematically investigated in isocratic HILIC-WAX separations of heparan sulfate disaccharides. The combined use of 75% acetonitrile with ammonium formate had overall benefits regarding intensity, detection limits, and peak shape for all salt concentrations investigated. Results obtained with the isocratic measurements suggested the potential use of a salt gradient method in order to maximize separation efficiency. A 3-step gradient from 14 mM to 65 mM ammonium formate concentration proved to be ideal for separation and quantitation. The LOD of the resulting method was 0.8-1.5 fmol for the individual disaccharides and the LOQ was between 2.5-5 fmol. Outstanding linearity could be observed up to 2 pmol. This novel combination provided sufficient sensitivity for disaccharide analysis, which was demonstrated by the analysis of heparan sulfate samples from porcine and bovine origin.

Widespread presence of bovine proteins in human cell lines.

HPLC-MS/MS analysis of various human cell lines shows the presence of a major amount of bovine protein contaminants. These likely originate from fetal bovine serum (FBS), typically used in cell cultures. If evaluated against a human protein database, on average 10% of the identified human proteins will be misleading (bovine proteins, but indicated as if they were human). Bovine contaminants therefore may cause major bias in proteomic studies of cell cultures, if not considered explicitly.

Site-specific N-glycosylation of HeLa cell glycoproteins.

We have characterized site-specific N-glycosylation of the HeLa cell line glycoproteins, using a complex workflow based on high and low energy tandem mass spectrometry of glycopeptides. The objective was to obtain highly reliable data on common glycoforms, so rigorous data evaluation was performed. The analysis revealed the presence of a high amount of bovine serum contaminants originating from the cell culture media - nearly 50% of all glycans were of bovine origin. Unaccounted, the presence of bovine serum components causes major bias in the human cellular glycosylation pattern; as is shown when literature results using released glycan analysis are compared. We have reliably identified 43 (human) glycoproteins, 69 N-glycosylation sites, and 178 glycoforms. HeLa glycoproteins were found to be highly (68.7%) fucosylated. A medium degree of sialylation was observed, on average 46.8% of possible sialylation sites were occupied. High-mannose sugars were expressed in large amounts, as expected in the case of a cancer cell line. Glycosylation in HeLa cells is highly variable. It is markedly different not only on various proteins but also at the different glycosylation sites of the same protein. Our method enabled the detailed characterization of site-specific N-glycosylation of several glycoproteins expressed in HeLa cell line.

Localized Amyloidosis of the Upper Aerodigestive Tract: Complex Analysis of the Cellular Infiltrate and the Amyloid Mass.

OBJECTIVES: The aim of this study was to analyse the composition of amyloid mass and the plasmacytic infiltrate of localized amyloidosis of the upper aerodigestive tract. METHODS: Biopsy materials were studied by light microscopy, immunohistochemistry (IHC), and mRNA in situ hybridization (mRNA-ISH). The amyloid mass was also analysed with high-performance liquid chromatography mass spectrometry- (HPLC-MS-) based proteomics. RESULTS: Nodular and diffuse forms of amyloid deposition were detected. IHC analysis revealed λ-light chain (LC) in two cases, κ-LC in one case. The remaining two were positive with both. Proteins, well known from other amyloidoses like amyloid A (AA), prealbumin/transthyretin (PA), apolipoprotein A-I (ApoAI), and amyloid P component (APC), and also keratin were found with variable intensities in the cases. HPLC-MS revealed dozens of proteins with both LCs in all the lesions but sometimes with surprisingly small intensities. mRNA-ISH analysis revealed identical λ and κ dominance and only one normal κ/λ cell ratio. CONCLUSION: Cellular infiltrate and protein components in the amyloid showed congruent results in all but one case. The only exception with normal cell ratio and λ-dominant amyloid could be originated from the different protein-secreting activity of plasma cell clones. HPLC-MS analysis explored both LCs in all the amyloid in variable amount, but other proteins with much higher intensities like keratins, apolipoprotein A-IV (ApoAIV), were also detected. Proteins like AA, PA, ApoAI, and APC, previously known about amyloid-forming capability, also appeared. This indicates that localized amyloid in the upper aerodigestive tract is not a homogenous immunoglobulin mass but a mixture of proteins. The sometimes very low light chain intensities might also suggest that not all the localized amyloidosis cases of the upper aerodigestive tract are of convincingly AL type, and the analysis of the cellular infiltrate might indicate that not all are monoclonal.

HDX and Native Mass Spectrometry Reveals the Different Structural Basis for Interaction of the Staphylococcal Pathogenicity Island Repressor Stl with Dimeric and Trimeric Phage dUTPases.

The dUTPase enzyme family plays an essential role in maintaining the genome integrity and are represented by two distinct classes of proteins; the ß-pleated homotrimeric and the all-α homodimeric dUTPases. Representatives of both trimeric and dimeric dUTPases are encoded by Staphylococcus aureus phage genomes and have been shown to interact with the Stl repressor protein of S. aureus pathogenicity island SaPIbov1. In the present work we set out to characterize the interactions between these proteins based on a range of biochemical and biophysical methods and shed light on the binding mechanism of the dimeric φNM1 phage dUTPase and Stl. Using hydrogen deuterium exchange mass spectrometry, we also characterize the protein regions involved in the dUTPase:Stl interactions. Based on these results we provide reasonable explanation for the enzyme inhibitory effect of Stl observed in both types of complexes. Our experiments reveal that Stl employs different peptide segments and stoichiometry for the two different phage dUTPases which allows us to propose a functional plasticity of Stl. The malleable character of Stl serves as a basis for the inhibition of both dimeric and trimeric dUTPases.

Identification of bifucosylated glycoforms using low-energy CID spectra.

We have used tandem mass spectrometry (MS/MS)-based analysis of glycopeptides in order to identify the composition and structure of rare glycoforms. The results illustrate utility of low-energy MS/MS for structure identification. We have shown the presence of bifucosylated and trifucosylated glycoforms in human α-1-acid glycoprotein (AGP), a major plasma glycoprotein. Fucosylation in the case of AGP always occurs on the antennae; core fucosylation was not observed.