Studies on Loading Salicylic Acid in Xerogel Films of Crosslinked Hyaluronic Acid.

During the last decades, salicylic acid (SA) and hyaluronic acid (HA) have been studiedfor a wide range of cosmetic and pharmaceutical applications. The current study investigated thedrug loading potential of SA in HA-based crosslinked hydrogel films using a post-loading (osmosis)method of the unmedicated xerogels from saturated aqueous solutions of salicylic acid over a rangeof pH values. The films were characterized with Fourier-transform infra-red spectroscopy (FT-IR) andultraviolet-visible (UV-Vis) spectrophotometry in order to elucidate the drug loading profile and thefilms’ integrity during the loading process. Additional studies on their weight loss (%), gel fraction(%), thickness increase (%) and swelling (%) were performed. Overall, the studies showed significantfilm disintegration at highly acidic and basic solutions. No drug loading occurred at neutral andbasic pH, possibly due to the anionic repulsion between SA and HA, whereas at, pH 2.1, the drugloading was promising and could be detected via UV-Vis analysis of the medicated solutions, withthe SA concentration in the xerogel films at 28% w/w

Studies on Loading Salicylic Acid in Xerogel Films of Crosslinked Hyaluronic Acid.

During the last decades, salicylic acid (SA) and hyaluronic acid (HA) have been studiedfor a wide range of cosmetic and pharmaceutical applications. The current study investigated thedrug loading potential of SA in HA-based crosslinked hydrogel films using a post-loading (osmosis)method of the unmedicated xerogels from saturated aqueous solutions of salicylic acid over a rangeof pH values. The films were characterized with Fourier-transform infra-red spectroscopy (FT-IR) andultraviolet-visible (UV-Vis) spectrophotometry in order to elucidate the drug loading profile and thefilms’ integrity during the loading process. Additional studies on their weight loss (%), gel fraction(%), thickness increase (%) and swelling (%) were performed. Overall, the studies showed significantfilm disintegration at highly acidic and basic solutions. No drug loading occurred at neutral andbasic pH, possibly due to the anionic repulsion between SA and HA, whereas at, pH 2.1, the drugloading was promising and could be detected via UV-Vis analysis of the medicated solutions, withthe SA concentration in the xerogel films at 28% w/w

Evaluation and Optimization of Mass Spectrometric Settings during Data-dependent Acquisition Mode: Focus on LTQ-Orbitrap Mass Analyzers

Mass-spectrometry-based proteomics has evolved as the preferred method for the analysis of complex proteomes. Undoubtedly, recent advances in mass spectrometry instrumentation have greatly enhanced proteomic analysis. A popular instrument platform in proteomics research is the LTQ-Orbitrap mass analyzer. In this tutorial, we discuss the significance of evaluating and optimizing mass spectrometric settings on the LTQ-Orbitrap during CID data-dependent acquisition (DDA) mode to improve protein and peptide identification rates. We focus on those MS and MS/MS parameters that have been systematically examined and evaluated by several researchers and are commonly used during DDA. More specifically, we discuss the effect of mass resolving power, preview mode for FTMS scan, monoisotopic precursor selection, signal threshold for triggering MS/MS events, number of microscans per MS/MS scan, number of MS/MS events, automatic gain control target value (ion population) for MS and MS/MS, maximum ion injection time for MS/MS, rapid and normal scan rate, and prediction of ion injection time. We furthermore present data from the latest generation LTQ-Orbitrap system, the Orbitrap Elite, along with recommended MS and MS/MS parameters. The Orbitrap Elite outperforms the Orbitrap Classic in terms of scan speed, sensitivity, dynamic range, and resolving power and results in higher identification rates. Several of the optimized MS parameters determined on the LTQ-Orbitrap Classic and XL were easily transferable to the Orbitrap Elite, whereas others needed to be reevaluated. Finally, the Q Exactive and HCD are briefly discussed, as well as sample preparation, LC-optimization, and bioinformatics analysis. We hope this tutorial will serve as guidance for researchers new to the field of proteomics and assist in achieving optimal results

Data-Dependent Middle-Down Nano-Liquid Chromatography–Electron Capture Dissociation-Tandem Mass Spectrometry: An Application for the Analysis of Unfractionated Histones

Middle-down mass spectrometry (MS) combined with electron capture dissociation (ECD) represents an attractive method for characterization of proteins and their post-translational modifications (PTMs). Coupling online chromatographic separation with tandem mass spectrometry enables a high-throughput analysis, while improving sensitivity of the electrosprayed peptides and reducing sample amount requirements. However, middle-down ECD has not been thus far coupled with online chromatographic separation. In this work, we examine the feasibility of coupling middle-down ECD with online nanoflow-liqiud chromatography (nano-LC) for the analysis of large, >3 kDa, and highly modified polypeptides in a data-dependent acquisition mode. We evaluate the effectiveness of the method by analyzing peptides derived from Asp-N and Glu-C digestions of unfractionated histones from calf thymus and acid-extracted histones from HeLa, MCF-7, and Jurkat cells. Our results demonstrate that middle-down ECD is compatible with online chromatographic separation, providing high peptide and protein sequence coverage while allowing precise mapping of PTM sites. The high mass accuracy, obtained by the ICR mass analyzer, for both the precursor and product ions greatly increases confidence in peptide identification, particularly for modified peptides. Overall, for all samples examined, several histone variants were identified and modification sites were successfully localized, including single, multiple, and positional isomeric PTM sites. The vast majority of the identified peptides were in the mass range from 3 to 9 kDa. The data presented here highlight the feasibility and utility of nano-LC–ECD-MS/MS for high-throughput middle-down analysis

The top-down, middle-down, and bottom-up mass spectrometry approaches for characterization of histone variants and their post-translational modifications

Epigenetic regulation of gene expression is, at least in part, mediated by histone modifications. PTMs of histones change chromatin structure and regulate gene transcription, DNA damage repair, and DNA replication. Thus, studying histone variants and their modifications not only elucidates their functional mechanisms in chromatin regulation, but also provides insights into phenotypes and diseases. A challenge in this field is to determine the best approach(es) to identify histone variants and their PTMs using a robust high-throughput analysis. The large number of histone variants and the enormous diversity that can be generated through combinatorial modifications, also known as histone code, makes identification of histone PTMs a laborious task. MS has been proven to be a powerful tool in this regard. Here, we focus on bottom-up, middle-down, and top-down MS approaches, including CID and electron-capture dissociation/electron-transfer dissociation based techniques for characterization of histones and their PTMs. In addition, we discuss advances in chromatographic separation that take advantage of the chemical properties of the specific histone modifications. This review is also unique in its discussion of current bioinformatic strategies for comprehensive histone code analysis

A cationic cysteine-hydrazide as an enrichment tool for the mass spectrometric characterization of bacterial free oligosaccharides

In Campylobacterales and related ε-proteobacteria with N-linked glycosylation (NLG) pathways, free oligosaccharides (fOS) are released into the periplasmic space from lipid-linked precursors by the bacterial oligosaccharyltransferase (PglB). This hydrolysis results in the same molecular structure as the oligosaccharide that is transferred to a protein to be glycosylated. This allowed for the general elucidation of the fOS-branched structures and monosaccharides from a number of species using standard enrichment and mass spectrometry methods. To aid characterization of fOS, hydrazide chemistry has often been used for chemical modification of the reducing part of oligosaccharides resulting in better selectivity and sensitivity in mass spectrometry; however, the removal of the unreacted reagents used for the modification often causes the loss of the sample. Here, we develop a more robust method for fOS purification and characterize glycostructures using complementary tandem mass spectrometry (MS/MS) analysis. A cationic cysteine hydrazide derivative was synthesized to selectively isolate fOS from periplasmic fractions of bacteria. The cysteine hydrazide nicotinamide (Cyhn) probe possesses both thiol and cationic moieties. The former enables reversible conjugation to a thiol-activated solid support, while the latter improves the ionization signal during MS analysis. This enrichment was validated on the well-studied Campylobacter jejuni by identifying fOS from the periplasmic extracts. Using complementary MS/MS analysis, we approximated data of a known structure of the fOS from Campylobacter concisus. This versatile enrichment technique allows for the exploration of a diversity of protein glycosylation pathways

Protein and Lantibiotic Sequencing by Gas Phase Dissociation Involving Vibrational Excitation and Ion Electron Reactions.

In proteomics, protein identification and characterization are largely performed by tandem mass spectrometry (MS/MS), in which sequence specific product ions are generated from precursor peptide or protein ions. Such MS/MS data can reveal the protein identity either by database search, or via de novo sequencing. However, successful and confident protein identification, either by database searching or by de novo sequencing, relies heavily on the extent and quality of the obtained sequence information generated by MS/MS. In addition, the higher the extent of fragmentation, the higher the probability of localizing post-translational modifications (PTMs). Electron based reactions, electron capture dissociation (ECD) and electron detachment dissociation (EDD), have shown great promise for PTM analysis, and for improved peptide sequence coverage. In this thesis, ion electron reactions, ECD, EDD, and electron induced dissociation (EID), are explored for peptide sequencing, and for PTM analysis. Disulfide bond formation is a PTM present in extracellular proteins. We demonstrate that EDD and infrared multiphoton dissociation (IRMPD) of peptide anions containing disulfide linkages result in preferential cleavage of S-S and C-S bonds and, therefore, both techniques can be used for probing disulfide bonds in peptide anions. Factors such as precursor ion charge state and m/z value, peptide mass, and protease selection that may influence the dissociation outcome in ECD were investigated, aiming to improve peptide sequence coverage. We show that doubly protonated peptides do not fragment efficiently in ECD, and that precursor ion m/z value is the main factor determining a successful ECD outcome. Highly charged precursor ions at m/z < ~960 fragment efficiently in ECD and yield high peptide sequence coverage. The utility of EID for dissociation of singly deprotonated species was also explored. We show that EID results in extensive fragmentation, providing structural information for peptide anions. For modified peptides, EID results in retention of sulfation and phosphorylation allowing localization of the modification site. Vibrational excitation, collision induced dissociation and IRMPD, were explored for structural characterization of native and oxidized lantibiotics. These experiments provided insights into the fragmentation behavior of native and oxidized lantibiotics, allowing prediction of their fragmentation pathways.Ph.D.ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/62221/1/kalliana_1.pd

Electron Capture Dissociation of Hydrogen- Deficient Peptide Radical Cations

Hydrogen-deficient peptide radical cations exhibit fascinating gas phase chemistry, which is governed by radical driven dissociation and, in many cases, by a combination of radical and charge driven fragmentation. Here we examine electron capture dissociation (ECD) of doubly, [M + H]^(2+•), and triply, [M + 2H]^(3+•), charged hydrogen-deficient species, aiming to investigate the effect of a hydrogen-deficient radical site on the ECD outcome and characterize the dissociation pathways of hydrogen-deficient species in ECD. ECD of [M + H]^(2+•) and [M + 2H]^(3+•) precursor ions resulted in efficient electron capture by the hydrogen-deficient species. However, the intensities of c- and z-type product ions were reduced, compared with those observed for the even electron species, indicating suppression of N–Cα backbone bond cleavages. We postulate that radical recombination occurs after the initial electron capture event leading to a stable even electron intermediate, which does not trigger N–Cα bond dissociations. Although the intensities of c- and z-type product ions were reduced, the number of backbone bond cleavages remained largely unaffected between the ECD spectra of the even electron and hydrogen-deficient species. We hypothesize that a small ion population exist as a biradical, which can trigger N–C_α bond cleavages. Alternatively, radical recombination and N–C_α bond cleavages can be in competition, with radical recombination being the dominant pathway and N–C_α cleavages occurring to a lesser degree. Formation of b- and y-type ions observed for two of the hydrogendeficient peptides examined is also discussed

Effect of mass spectrometric parameters on peptide and protein identification rates for shotgun proteomic experiments on an LTQ-orbitrap mass analyzer

The success of a shotgun proteomic experiment relies heavily on the performance and optimization of both the LC and the MS systems. Despite this, little consideration has, so far, been given to the importance of evaluating and optimizing the MS instrument settings during data-dependent acquisition mode. Moreover, during data-dependent acquisition, the users have to decide and choose among various MS parameters and settings, making a successful analysis even more challenging. We have systematically investigated and evaluated the effect of enabling and disabling the preview mode for FTMS scan, the number of microscans per MS/MS scan, the number of MS/MS events, the maximum ion injection time for MS/MS, and the automatic gain control target value for MS and MS/MS events on protein and peptide identification rates on an LTQ-Orbitrap using the Saccharomyces cerevisiae proteome. Our investigations aimed to assess the significance of each MS parameter to improve proteome analysis and coverage. We observed that higher identification rates were obtained at lower ion injection times i.e. 50–150 ms, by performing one microscan and 12–15 MS/MS events. In terms of ion population, optimal automatic gain control target values were at 5×10^5–1×10^6 ions for MS and 3×10^3–1×10^4 ions for MS/MS. The preview mode scan had a minimal effect on identification rates. Using optimized MS settings, we identified 1038 (±2.3%) protein groups with a minimum of two peptide identifications and an estimated false discovery rate of ∼1% at both peptide and protein level in a 160-min LC-MS/MS analysis