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).

Synthesis and Anticancer Activity of Phosphinoylated and Phosphonoylated N-Heterocycles Obtained by the Microwave-Assisted Palladium Acetate-Catalyzed Hirao Reaction.

A literature survey showed that different derivatives with the 9-phenyl-9H-carbazole or the dihydroindoline scaffold may be of biological activity including cytotoxic effect. Driven by this experience, P-functionalized derivatives of these N-heterocycles were synthesized. Three N-heterocycles, 9-(4-bromophenyl)-9H-carbazole, 3-bromo-9-phenyl-9H-carbazole and 1-(5-bromoindolin-1-yl)ethan-1-one, were coupled with dialkyl phosphites and diarylphosphine oxides using Pd(OAc)2 (10 %) as the catalyst precursor and triethylamine as the base in ethanol under microwave irradiation. The excess of the Y2 P(O)H reagent (Y=alkoxy, aryl) (30 %) served as the P-ligand in its trivalent tautomeric form (Y2 POH), hence there was no need for the usual P-ligands meaning cost and environmental burden. Hence, the presented method is a "green" approach that proved to be more efficient than the preparation by the traditional method. The products, dialkyl phosphonates and tertiary phosphine oxides obtained in 58-84 % yields were characterized, one of them also by single crystal X-ray analysis, and were subjected to in vitro biological activity evaluation. A (carbazol)yl-phenylphosphonate, an N-phenyl-(carbazol)yl-phosphonate, a (carbazol)yl-phenylphosphine oxide and an N-phenyl-(carbazol)ylphosphine oxide revealed a significant cytotoxic activity on A549 human non-small-cell lung carcinoma and MonoMac-6 acute monocytic leukemia cancer cells. The cytotoxic effect was significant as compared to that of the reference compounds.

A Study of the Bisphosphonic Derivatives from the Pudovik Reaction of Dialkyl α-Oxophosphonates and >P(O)H Reagents: X-ray Structure and Bioactivity.

New hydroxy-methylenebisphosphonic derivatives were prepared with different P-functions. The outcome of the reaction of α-oxophosphonates (YC(O)P(O)(OR)2) and dialkyl phosphites or diarylphosphine oxides depended on the Y substituent of the oxo-compound, the nature of the P-reagent and the amount of the diethylamine catalyst. Starting from dimethyl α-oxoethylphosphonate, in the presence of 5% of diethylamine, the corresponding Pudovik adduct was the single product. While using 40% of the catalyst, the rearranged species with the >P(O)-O-CH-P(O)< skeleton was the exclusive component. A similar reaction of α-oxobenzylphosphonate followed the rearrangement protocol. X-ray crystallography revealed not only the spatial structures of the three products, but also an intricate pattern evolving from the interplay of slight chemical differences, solvent inclusion and disorder as well as H-bridge patterns, which invite further investigation. In vitro activity of the compounds was assessed on different tumor cell cultures using end-point-type cell tetrazolium-based measurements. These structure-activity studies revealed a cytostatic effect for four rearranged derivatives containing aromatic units. One of them had a pronounced effect on MDA-MB 231 and Ebc-1 cells, showing IC50 = 37.8 and 25.9 µM, respectively.

Optimization of reversed-phase solid-phase extraction for shotgun proteomics analysis.

Reversed-phase solid-phase extraction (SPE) is the method of choice for the purification of proteomics samples. Even though the efficacy of SPE methods is sample type-dependent, the manufacturers' protocols are used in most studies. Using an optimized SPE method can lead to a substantial gain in identification and recovery. In this tutorial, we give a brief introduction to the most important parameters influencing SPE performance, and we present a short workflow (16 measurements) for optimizing the SPE procedure. This is complemented by method performance assessment instructions and a short troubleshooting guide to help users further understand and investigate their SPE methods.

Compositional Analysis of Glycosaminoglycans in Different Lung Cancer Types-A Pilot Study.

Lung cancer is one of the most commonly diagnosed cancer types. Studying the molecular changes that occur in lung cancer is important to understand tumor formation and identify new therapeutic targets and early markers of the disease to decrease mortality. Glycosaminoglycan chains play important roles in various signaling events in the tumor microenvironment. Therefore, we have determined the quantity and sulfation characteristics of chondroitin sulfate and heparan sulfate in formalin-fixed paraffin-embedded human lung tissue samples belonging to different lung cancer types as well as tumor adjacent normal areas. Glycosaminoglycan disaccharide analysis was performed using HPLC-MS following on-surface lyase digestion. Significant changes were identified predominantly in the case of chondroitin sulfate; for example, the total amount was higher in tumor tissue compared to the adjacent normal tissue. We also observed differences in the degree of sulfation and relative proportions of individual chondroitin sulfate disaccharides between lung cancer types and adjacent normal tissue. Furthermore, the differences in the 6-O-/4-O-sulfation ratio of chondroitin sulfate were different between the lung cancer types. Our pilot study revealed that further investigation of the role of chondroitin sulfate chains and enzymes involved in their biosynthesis is an important aspect of lung cancer research.

Inter- and intratumoral proteomics and glycosaminoglycan characterization of ALK rearranged lung adenocarcinoma tissues: a pilot study.

Lung cancer is one of the most common types of cancer with limited therapeutic options, therefore a detailed understanding of the underlying molecular changes is of utmost importance. In this pilot study, we investigated the proteomic and glycosaminoglycan (GAG) profile of ALK rearranged lung tumor tissue regions based on the morphological classification, mucin and stromal content. Principal component analysis and hierarchical clustering revealed that both the proteomic and GAG-omic profiles are highly dependent on mucin content and to a lesser extent on morphology. We found that differentially expressed proteins between morphologically different tumor types are primarily involved in the regulation of protein synthesis, whereas those between adjacent normal and different tumor regions take part in several other biological processes (e.g. extracellular matrix organization, oxidation-reduction processes, protein folding) as well. The total amount and the sulfation profile of heparan sulfate and chondroitin sulfate showed small differences based on morphology and larger differences based on mucin content of the tumor, while an increase was observed in both the total amount and the average rate of sulfation in tumors compared to adjacent normal regions.

Activation-Free Sulfonyl Fluoride Probes for Fragment Screening.

SuFEx chemistry is based on the unique reactivity of the sulfonyl fluoride group with a range of nucleophiles. Accordingly, sulfonyl fluorides label multiple nucleophilic amino acid residues, making these reagents popular in both chemical biology and medicinal chemistry applications. The reactivity of sulfonyl fluorides nominates this warhead chemotype as a candidate for an external, activation-free general labelling tag. Here, we report the synthesis and characterization of a small sulfonyl fluoride library that yielded the 3-carboxybenzenesulfonyl fluoride warhead for tagging tractable targets at nucleophilic residues. Based on these results, we propose that coupling diverse fragments to this warhead would result in a library of sulfonyl fluoride bits (SuFBits), available for screening against protein targets. SuFBits will label the target if it binds to the core fragment, which facilitates the identification of weak fragments by mass spectrometry.

New N-acyl- as well as N-phosphonoylmethyl- and N-phosphinoylmethyl-α-amino-benzylphosphonates by acylation and a tandem Kabachnik-Fields protocol.

Diethyl α-benzylamino- and α-amino-benzylphosphonates obtained by the Kabachnik-Fields reaction were useful intermediates in the synthesis of other derivatives. Acylation of α-aminophosphonates with acyl chlorides led to the corresponding N-acyl species existing under a dynamic equilibrium of two conformers. Judging from the broad NMR signals, the sterically most crowded N-benzoyl-N-benzyl derivative suffered a hindered rotation around the N-C axis to the acyl carbon atom at 26 °C. Low temperature NMR measurements at -10 °C showed the presence of two distinct rotamers that were characterized. The other acylated α-amino-benzylphosphonates prepared revealed a less hindered rotation. Single crystal X-ray diffraction of the NH-propionyl species showed a dimer, in which the two molecules were held together by rare intermolecular PO⋯HN bonds. On the other hand, substituted α-benzylamino-benzylphosphonates prepared by phospha-Mannich reactions were employed, as a new approach, in a second Kabachnik-Fields condensation by reaction with formaldehyde and dialkyl phosphites or secondary phosphine oxides to afford novel N-phosphonoylmethyl- and N-phosphinoylmethyl-α-amino-benzylphosphonates. The structure of the new products was confirmed by two-dimensional NMR spectroscopy. A symmetrical bis derivative was prepared in a diastereoselective manner. A related tris(phosphonoylmethyl)amine species was also synthesized.

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.

Synthesis of Phosphonates with Identical and Different Alkyl Groups by the Microwave-Assisted Alkylation of Phosphonic Ester-Acid Derivatives.

Monoalkyl phosphonic derivatives obtained by the microwave (MW)- and ionic liquid-promoted direct esterification of alkylphosphonic acids were converted to the corresponding dialkyl alkylphosphonates on reaction with alkyl halides in the presence of triethylamine, under solvent-free MW-assisted conditions. Derivatives with different alkoxy groups were also synthesized. A minor "disproportionation" side reaction was identified during the preparation of dialkyl alkylphosphonates with different alkoxy groups. All together 12 alkylphosphonates were prepared by the efficient method developed.

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.

Comparison of solid-phase extraction methods for efficient purification of phosphopeptides with low sample amounts.

Efficient phosphoproteomic analysis of small amounts of biological samples (e.g. tissue biopsies) requires carefully selected enrichment and purification steps prior to the nanoflow HPLC-MS/MS analysis. Solid-phase extraction (SPE) is one of the most commonly used approaches for sample preparation. Several stationary phases are available for peptide SPE purification, however, most of the published methods are not optimized to provide good recoveries of phosphorylated peptides. Our goal was to investigate the performance of 13 self-packed and 3 commercial centrifugal SPE cartridges/spin tips, thus enhancing the efficiency of the phosphoproteomic analysis of small amounts of complex protein mixtures. Eight reversed-phase (RP), five graphite, two ion-exchange, and one hydrophilic-lipophilic balance (HLB) stationary phase were evaluated. Two RP, one graphite, and the HLB self-packed centrifugal SPE tips provided excellent results for the purification of 1 µg tissue and cell line digests. Using these methods, the sample loss was significantly reduced compared to one of the commercial SPE methods, 22-58% more unique phosphopeptides were identified, and the recovery was higher by 132-155%.

Glycosaminoglycan Analysis of FFPE Tissues from Prostate Cancer and Benign Prostate Hyperplasia Patients Reveals Altered Regulatory Functions and Independent Markers for Survival.

Prostate cancer is one of the most frequent cancer types among men. Several biomarkers and risk assessment methods are already available; however, enhancing their selectivity and sensitivity is still necessary. For improving therapeutic decisions, both basic and clinical research studies are still ongoing for a better understanding of the underlying molecular mechanisms. The enzymatic digests of heparan sulfate (HS) and chondroitin sulfate (CS) chains were investigated in tissue samples taken from patients with prostate cancer (PCa) and benign prostate hyperplasia (BPH) with the HPLC-MS methodology. None of the HS species analyzed showed correlating alterations with currently used markers such as clinical stage, Gleason score, or prostate-specific antigen (PSA) level. The total quantity and sulfation motifs of CS were both significantly different among BPH and different risk groups of PCa. Furthermore, the cancer-specific survival of patients can be predicted based on the levels of non-sulfated and doubly sulfated CS disaccharides as well as the total HS content and the doubly and triply sulfated HS disaccharide ratios. These disaccharide ratios proved to be independent markers from clinical parameters. Further investigations of glycosaminoglycan motifs were proposed for the validation of the results on independent patient cohorts as well.

Optimized Sample Preparation and Microscale Separation Methods for High-Sensitivity Analysis of Hydrophilic Peptides.

The optimization of solid-phase extraction (SPE) purification and chromatographic separation is usually neglected during proteomics studies. However, the effects on detection performance are not negligible, especially when working with highly glycosylated samples. We performed a comparative study of different SPE setups, including an in-house optimized method and reversed-phase chromatographic gradients for the analysis of highly glycosylated plasma fractions as a model sample for glycopeptide analysis. The in-house-developed SPE method outperformed the graphite-based and hydrophilic interaction liquid chromatography (HILIC) purification methods in detection performance, recovery, and repeatability. During optimization of the chromatography, peak distribution was maximized to increase the peptide detection rate. As a result, we present sample purification and chromatographic separation methods optimized for the analysis of hydrophilic samples, the most important of which is heavily N-glycosylated protein mixtures.

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).

Proteomic Changes of Osteoclast Differentiation in Rheumatoid and Psoriatic Arthritis Reveal Functional Differences.

Background: Osteoclasts play a crucial role in the maintenance, repair, and remodeling of bones of the adult vertebral skeleton due to their bone resorption capability. Rheumatoid arthritis (RA) and psoriatic arthritis (PsA) are associated with increased activity of osteoclasts. Objectives: Our study aimed to investigate the dynamic proteomic changes during osteoclast differentiation in healthy donors, in RA, and PsA. Methods: Blood samples of healthy donors, RA, and PsA patients were collected, and monocytes were isolated and differentiated into osteoclasts in vitro using macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANK-L). Mass spectrometry-based proteomics was used to analyze proteins from cell lysates. The expression changes were analyzed with Gene Set Enrichment Analysis (GSEA). Results: The analysis of the proteomic changes revealed that during the differentiation of the human osteoclasts, expression of the proteins involved in metabolic activity, secretory function, and cell polarity is increased; by contrast, signaling pathways involved in the immune functions are downregulated. Interestingly, the differences between cells of healthy donors and RA/PsA patients are most pronounced after the final steps of differentiation to osteoclasts. In addition, both in RA and PsA the differentiation is characterized by decreased metabolic activity, associated with various immune pathway activities; furthermore by accelerated cytokine production in RA. Conclusions: Our results shed light on the characteristic proteomic changes during human osteoclast differentiation and expression differences in RA and PsA, which reveal important pathophysiological insights in both diseases.

A Study on the Direct Esterification of Monoalkylphosphates and Dialkylphosphates; The Conversion of the Latter Species to Trialkylphosphates by Alkylating Esterification.

The microwave (MW)-assisted direct esterification of certain P-acids is a green method. Quantum chemical calculations revealed that the activation enthalpy (ΔH#) for the exothermic monoalkylphosphate → dialkylphosphate transformation was on the average 156.6 kJ mol-1, while ΔH# for the dialkylphosphate → trialkylphosphate conversion was somewhat higher, 171.2 kJ mol-1, and the energetics of the elemental steps of this esterification was less favorable. The direct monoesterification may be performed on MW irradiation in the presence of a suitable ionic liquid additive. However, the second step, with the less favorable energetics as a whole, could not be promoted by MWs. Hence, dialkylphosphates had to be converted to triesters by another method that was alkylation. In this way, it was also possible to synthesize triesters with different alkyl groups. Eventually a green, P-chloride free MW-promoted two-step method was elaborated for the synthesis of phosphate triesters.