This study demonstrates how the latest ultrahigh-performance liquid chromatography (UHPLC) technology can be combined with high-resolution accurate-mass (HRAM) mass spectrometry (MS) and long columns packed with fully porous particles to improve bottom-up proteomics analysis with nanoflow liquid chromatography–mass spectrometry (nanoLC-MS) methods. The increased back pressures from the UHPLC system enabled the use of 75 μm I.D. × 75 cm columns packed with 2 μm particles at a typical 300 nL/min flow rate as well as elevated and reduced flow rates. The constant pressure pump operation at 1500 bar reduced sample loading and column washing/equilibration stages and overall overhead time, which maximizes MS utilization time. The versatility of flow rate optimization to balance the sensitivity, throughput with sample loading amount, and capability of using longer gradients contributes to a greater number of peptide and protein identifications for single-shot bottom-up proteomics experiments. The routine proteome profiling and precise quantification of >7000 proteins with single-shot nanoLC-MS analysis open possibilities for large-scale discovery studies with a deep dive into the protein level alterations. Data are available via ProteomeXchange with identifier PXD035665.

ABSTRACT In the field of LC-MS based proteomics, increases in sampling depth and proteome coverage have mainly been accomplished by rapid advances in mass spectrometer technology. The comprehensiveness and quality of data that can be generated do however also depend on the performance provided by nano liquid chromatography (nanoLC) separations. Proper selection of reversed-phase separation columns can be important to provide the MS instrument with peptides at the highest possible concentration and separated at the highest possible resolution. In the current contribution, we evaluate the use of prototype generation 2 μPAC nanoLC columns which use C18 functionalized superficially porous micro pillars as a stationary phase. When comparing to traditionally used fully porous silica stationary phases, more precursors could be characterized when performing single shot data-dependent LC-MS/MS analyses of a human cell line tryptic digest. Up to 30% more protein groups and 60% more unique peptides were identified for short gradients (10 min) and limited sample amounts (10-100 ng of cell lysate digest). With LC-MS gradient times of 10, 60, 120 and 180 min, we respectively identified 2252, 6513, 7382 and 8174 protein groups with 25, 500, 1000 and 2000 ng of sample loaded on column. Reduction of sample carry over to the next run (up to 2 to 3%) and decreased levels of methionine oxidation (up to 3-fold) were identified as additional figures of merit. When analyzing a disuccinimidyl dibutyric urea (DSBU) crosslinked synthetic library, 29 to 59 more unique crosslinked peptides could be identified at a experimentally validated false discovery rate (FDR) of 1-2%.

Introduction Peptide analysis with nano-flow gradient separations on long (≥25 cm) analytical columns coupled with HRAM (high-resolution accurate-mass) mass spectrometry is the standard for discoverybased “bottom-up” proteomics research. While delivering unsurpassed sensitivity and depth of analysis, nano-flow setups have yet to achieve the throughput and robustness required for large sample cohort analysis. Therefore, nano-flow LC-MS has been of limited use for translational proteomics, in particular for validation studies.

A current trend in proteomics is to acquire data in a "single-shot" by LC-MS/MS because it simplifies workflows and promises better throughput and quantitative accuracy than schemes that involve extensive sample fractionation. However, single-shot approaches can suffer from limited proteome coverage when performed by data dependent acquisition (ssDDA) on nanoflow LC systems. For applications where sample quantities are not scarce, this study shows that high proteome coverage can be obtained using a microflow LC-MS/MS system operating a 1 mm i.d. × 150 mm column, at a flow-rate of 50 μL/min and coupled to an Orbitrap HF-X mass spectrometer. The results demonstrate the identification of ∼9 000 proteins from 50 μg of protein digest from Arabidopsis roots, 7 500 from mouse thymus, and 7 300 from human breast cancer cells in 3 h of analysis time in a single run. The dynamic range of protein quantification measured by the iBAQ approach spanned 5 orders of magnitude and replicate analysis showed that the median coefficient of variation was below 20%. Together, this study shows that ssDDA by μLC-MS/MS is a robust method for comprehensive and large-scale proteome analysis and which may be further extended to more rapid chromatography and data independent acquisition approaches in the future.̀.

Introduction Nano-flow LC-MS analysis employing long columns and gradients at nano-flow rates has long been established as the method of choice for bottom-up, discovery proteomics. Nevertheless, the challenges associated with nano-flow LC have hitherto limited its application to the field of academic research. The main concerns associated with the technique include locating and eliminating the sources of dead volume, leaks, and capillary or column blockages, combined with the struggle to generate reproducible data. Together, such obstacles can result in the loss of precious time and irreplaceable samples. Another caveat of nanoLC-MS applications is their limited sample throughput and MS utilization. Lower flow rates result in proportionally long periods of MS idle time due to time-consuming sample loading and column equilibration particularly when long separation columns are employed. The latest low-flow UHPLC systems and columns have the potential to overcome many of these challenges and to usher in a new era of robustness and standardization in the field of lowflow LC-MS based analytics.

Although current LC-MS technology permits scientists to efficiently screen clinical samples in translational research, e.g., steroids, biogenic amines, and even plasma or serum proteomes, in a daily routine, maintaining the balance between throughput and analytical depth is still a limiting factor. A typical approach to enhance the proteome depth is employing offline two-dimensional (2D) fractionation techniques before reversed-phase nanoLC-MS/MS analysis (1D-nanoLC-MS). These additional sample preparation steps usually require extensive sample manipulation, which could result in sample alteration and sample loss. Here, we present and compare 1D-nanoLC-MS with an automated online-2D high-pH RP × low pH RP separation method for deep proteome profiling using a nanoLC system coupled to a high-resolution accurate-mass mass spectrometer. The proof-of-principle study permitted the identification of ca. 500 proteins with ∼10,000 peptides in 15 enzymatically digested crude serum samples collected from healthy donors in 3 laboratories across Europe. The developed method identified 60% more peptides in comparison with conventional 1D nanoLC-MS/MS analysis with ca. 4 times lower throughput while retaining the quantitative information. Serum sample preparation related changes were revealed by applying unsupervised classification techniques and, therefore, must be taken into account while planning multicentric biomarker discovery and validation studies. Overall, this novel method reduces sample complexity and boosts the number of peptide and protein identifications without the need for extra sample handling procedures for samples equivalent to less than 1 μL of blood, which expands the space for potential biomarker discovery by looking deeper into the composition of biofluids.

This review gives an overview of current screening practices for cervical cancer. In the introduction, we will cover approaches of population screening focusing on high-risk Human Papilloma Virus (hrHPV) and the need for a better triage assay. We will further assess the impact of current vaccination programs on screening. Subsequently, the review will cover various technological aspects of nucleic acid- and protein-based biomarker assays. We will then detail different molecular markers in view of their use in triage assays, emphasizing epigenetic and protein markers. Finally, we will place this in the context of cost-effectiveness considerations in view of their implementation in high- as well as in low- to middle-income countries. Introduction Cervical cancer remains a significant healthcare problem, notably in low- to middle-income countries. While a negative test for hrHPV has a predictive value of more than 99.5%, its positive predictive value is less than 10% for CIN2+ stages. This makes the use of a so-called triage test indispensable for population-based screening to avoid referring women, that are ultimately at low risk of developing cervical cancer, to a gynecologist. This review will give an overview of tests that are based on epigenetic marker panels and protein markers. Areas covered There is a medical need for molecular markers with a better predictive value to discriminate hrHPV-positive women that are at risk of developing cervical cancer from those that are not. Areas covered are epigenetic and protein markers as well as health economic considerations in view of the fact that most cases of cervical cancer arise in low-to-middle-income countries. Expert opinion While there are biomarker assays based on changes at the nucleic acid (DNA methylation patterns, miRNAs) and at the protein level, they are not widely used in population screening. Combining nucleic acid-based and protein-based tests could improve the overall specificity for discriminating CIN2+ lesions that carry a low risk of progressing to cervical cancer within the screening interval from those that carry an elevated risk. The challenge is to reduce unnecessary referrals without an undesired increase in false-negative diagnoses resulting in cases of cervical cancer that could have been prevented. A further challenge is to develop tests for low-and middle-income countries, which is critical to reduce the worldwide burden of cervical cancer.

ABSTRACT This review gives an overview of current screening practices for cervical cancer. In the introduction, we will cover approaches of population screening focusing on high-risk Human Papilloma Virus (hrHPV) and the need for a better triage assay. We will further assess the impact of current vaccination programs on screening. Subsequently, the review will cover various technological aspects of nucleic acid- and protein-based biomarker assays. We will then detail different molecular markers in view of their use in triage assays, emphasizing epigenetic and protein markers. Finally, we will place this in the context of cost-effectiveness considerations in view of their implementation in high- as well as in low- to middle-income countries. Introduction Cervical cancer remains a significant healthcare problem, notably in low- to middle-income countries. While a negative test for hrHPV has a predictive value of more than 99.5%, its positive predictive value is less than 10% for CIN2+ stages. This makes the use of a so-called triage test indispensable for population-based screening to avoid referring women, that are ultimately at low risk of developing cervical cancer, to a gynecologist. This review will give an overview of tests that are based on epigenetic marker panels and protein markers. Areas covered There is a medical need for molecular markers with a better predictive value to discriminate hrHPV-positive women that are at risk of developing cervical cancer from those that are not. Areas covered are epigenetic and protein markers as well as health economic considerations in view of the fact that most cases of cervical cancer arise in low-to-middle-income countries. Expert opinion While there are biomarker assays based on changes at the nucleic acid (DNA methylation patterns, miRNAs) and at the protein level, they are not widely used in population screening. Combining nucleic acid-based and protein-based tests could improve the overall specificity for discriminating CIN2+ lesions that carry a low risk of progressing to cervical cancer within the screening interval from those that carry an elevated risk. The challenge is to reduce unnecessary referrals without an undesired increase in false-negative diagnoses resulting in cases of cervical cancer that could have been prevented. A further challenge is to develop tests for low-and middle-income countries, which is critical to reduce the worldwide burden of cervical cancer.

Lysine acetylations are post-translational modifications of cellular proteins, that are crucial in the regulation of many cellular processes. Lysine acetylations on histone proteins are part of the epigenetic code regulating gene expression and are installed by histone acetyltransferases. Observations that inflammatory lung diseases, such as asthma and chronic obstructive pulmonary disease, are characterized by increased histone acetyltransferase activity indicate that development of small molecule inhibitors for these enzymes might be a valuable approach towards new therapies for these diseases. The 6-alkylsalicylate MG149 is a candidate to explore this hypothesis because it has been demonstrated to inhibit the MYST type histone acetyltransferases. In this study, we determined the K-i value for inhibition of the MYST type histone acetyltransferase KAT8 by MG149 to be 39 +/- 7.7 mu M. Upon investigating whether the inhibition of histone acetyltransferases by MG149 correlates with inhibition of histone acetylation in murine precision-cut lung slices, inhibition of acetylation was observed using an LC-MS/ MS based assay on histone H4 res 4-17, which contains the target lysine of KAT8. Following up on this, upon treatment with MG149, reduced pro-inflammatory gene expression was observed in lipopolysaccharide and interferon gamma stimulated murine precision-cut lung slices. Based on this, we propose that 6-alkylsalicylates such as MG149 have potential for development towards applications in the treatment of inflammatory lung diseases. (C) 2017 Elsevier Ltd. All rights reserved.

Purpose The objective of this study is to better understand factors governing the variability and sensitivity in SRM and PRM, compared to immunoassay. Experimental design A 2D‐LC–MS/MS‐based SRM and PRM assay is developed for quantitative measurements of HSP90α in serum. Forty‐three control sera are compared by SRM, PRM, and ELISA following the manufacturer's instructions. Serum samples are trypsin‐digested and fractionated by strong cation exchange chromatography prior to SRM and PRM measurements. Analytical parameters such as linearity, LOD, LOQ, repeatability, and reproducibility of the SRM, PRM, and ELISA are determined. Results PRM data obtained by high‐resolution MS correlate better with ELISA measurements than SRM data measured on a triple quadrupole mass spectrometer. While all three methods (SRM, PRM, and ELISA) are able to quantify HSP90α in serum at the ng mL –1 level, the use of PRM on a high‐resolution mass spectrometer reduces variation and shows comparable sensitivity to immunoassay. Conclusions and clinical relevance Using fractionation, it is possible to measure ng mL –1 levels of HSP90α in a reproducible, selective, and sensitive way using PRM in serum. This opens up the possibility to use PRM in a multiplexed way as an attractive alternative for immunoassays without the use of antibodies or comparable binders.

The detection of very low concentrations of antibodies in complex matrices poses a large challenge in bioanalysis. The applied detection methods require high sensitivity and selectivity. Additionally, they need to be robust and fast in order to provide consistent data quality and high sample throughput. We developed a capillary flow LC-MS method which overcomes the sensitivity limitation of analytical flow setups and the throughput limitation of nano flow separations. Chromatographic separation was achieved using a Thermo ScientificTM UltiMateTM 3000 RSLCnano system in capillary flow configuration and 300 μm inner diameter AcclaimTM PepMap column. The UHPLC system was set-up in pre-concentration mode exploiting its micro flow pump for fast sample loading. Capillary flow LC together with high-resolution accurate-mass (HRAM) provided by the Thermo ScientificTM Q ExactiveTM HF mass spectrometer allows robust, high throughput detection of antibody with amol sensitivity and is around 20 times more sensitive in comparison with analytical flow LC-MS analysis.

We developed a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the site-specific quantification of lysine acetylation in the N-terminal region of histone H4 by combining chemical derivatization at the protein and peptide levels with digestion using chymotrypsin and trypsin. Unmodified ε-amino groups were first modified with propionic acid anhydride and the derivatized protein digested with trypsin and chymotrypsin. The newly formed peptide N-termini were subjected to a second derivatization step with d6- (heavy) or d0- (light) acetic acid anhydride. Samples were mixed at different ratios and peptides monitored by multiple reaction monitoring (MRM) LC-MS/MS. The method was validated in terms of linearity (R 2 ≥ 0.94), precision (RSD ≤ 10 %), and accuracy (≤27 %) and used to assess the effect of the histone deacetylase (HDAC) inhibitors SAHA and MS-275 in the murine macrophage-like cell line RAW 264.7. SAHA and MS-275 showed site-specific effects on the acetylation levels of K5 and K8 with the K5(Ac)–K8 and K5–K8(Ac) peptides increasing 2.5-fold and 5-fold upon treatment with SAHA and MS-275, respectively. Assessing lysine acetylation in a site-specific manner is important for gaining a better understanding of the effects of HDAC inhibitors and for clarifying disease mechanisms where lysine acetylation plays a role.

A micellar thin-layer chromatography (TLC) method has been developed and validated for the determination of gamma-aminobutyric acid (GABA) in pharmaceutical formulation "Aminalon" (coated tablets). Aqueous micellar mobile phase contained 0.005 mol.L-1 of non-ionic surfactant Brij-35, adjusted to pH 2.0 with hydrochloric acid was used for GABA separation. The reaction with ninhydrin was applied for visualization of chromatographic spots. The developed micellar TLC method allows assaying GABA in 20 min. The method was validated with respect to linearity, specificity, accuracy, precision and robustness. GABA response showed good linearity in the range of 240-360 mu g-mL(-1), LOD and LOQ were 8.3 mu g-mL(-1) and 25 mu g-mL(-1), respectively. The average %RSD value did not exceed 1.0%. The method is accurate, reproducible, cost-effective, and can be used in routine quality control analysis and for stability-indicating analysis.

Lysine acetylations are reversible posttranslational modifications of histone and non-histone proteins that play important regulatory roles in signal transduction cascades and gene expression. Lysine acetylations are regulated by histone acetyltransferases as writers and histone deacetylases as erasers. Because of their role in signal transduction cascades, these enzymes are important players in inflammation. Therefore, histone acetyltransferase inhibitors could reduce inflammatory responses. Among the few histone acetyltransferase inhibitors described, C646 is one of the most potent (Ki of 0.4μM for histone acetyltransferase p300). C646 was described to affect the NF-κB pathway; an important pathway in inflammatory responses, which is regulated by acetylation. This pathway has been implicated in asthma and COPD. Therefore, we hypothesized that via regulation of the NF-κB signaling pathway, C646 can inhibit pro-inflammatory gene expression, and have potential for the treatment of inflammatory lung diseases. In line with this, we demonstrate here that C646 reduces pro-inflammatory gene expression in RAW264.7 murine macrophages and murine precision-cut lung slices. To unravel its effects on cellular substrates we applied mass spectrometry and found, counterintuitively, a slight increase in acetylation of histone H3. Based on this finding, and structural features of C646, we presumed inhibitory activity of C646 on histone deacetylases, and indeed found inhibition of histone deacetylases from 7μM and higher concentrations. This indicates that C646 has potential for further development towards applications in the treatment of inflammation, however, its newly discovered lack of selectivity at higher concentrations needs to be taken into account.

EVALUATION OF RUGGED AND OPTIMAL ANALYSİS CONDİTİONS İN MICELLAR LIOUID CHROMATOGRAPHY BY USİNG INTERPRETATIVE OPTIMIZATION STRATEGY AND DERRINGER’S DESIRABILITY FUNCTION Alexander P. Boichenko, Lidia P. Loginova, Artem U. Kulikov, E. Yu. Yakovleva Department of Chemical Metrology, V.N. Karazin Kharkov National University, Svoboda square, 4, 61077 Kharkov, Ukraine, +38(057)7075605 E-mail: boichenko@univer.kharkov.ua

The classification of chromatographic columns with bonded octadecyl groups in conventional reversed-phase high-performance liquid chromatography (RP-HPLC) and in micellar liquid chromatography (MLC) was completed using unsupervised pattern recognition methods: principal component analysis, cluster analysis, and self-organized Kohonen neural networks (KNN). KNN provided an unbiased classification of the chromatographic columns and can be used as a powerful tool for columns selection in routine analysis. Despite the covering of C18 bonded silica surface of stationary phase with surfactant monomers in MLC, which strongly affect the compounds retention, the selectivity of the test compounds separation in MLC as well as in conventional RP-HPLC is affected by the sorbent properties. Thus, elucidation of columns similarity in both HPLC modes is of primary importance for developing robust analytical methods and selection of the analogous sorbents.

We developed a discovery-validation mass-spectrometry-based pipeline to identify a set of proteins that are regulated in serum of patients with cervical intraepithelial neoplasia (CIN) and squamous cell cervical cancer using iTRAQ, label-free shotgun, and targeted mass-spectrometric quantification. In the discovery stage we used a "pooling" strategy for the comparative analysis of immunodepleted serum and revealed 15 up- and 26 down-regulated proteins in patients with early- (CES) and late-stage (CLS) cervical cancer. The analysis of nondepleted serum samples from patients with CIN, CES, an CLS and healthy controls showed significant changes in abundance of alpha-1-acid glycoprotein 1, alpha-1-antitrypsin, serotransferrin, haptoglobin, alpha-2-HS-glycoprotein, and vitamin D-binding protein. We validated our findings using a fast UHPLC/MRM method in an independent set of serum samples from patients with cervical cancer or CIN and healthy controls as well as serum samples from patients with ovarian cancer (more than 400 samples in total). The panel of six proteins showed 67% sensitivity and 88% specificity for discrimination of patients with CIN from healthy controls, a stage of the disease where current protein-based biomarkers, for example, squamous cell carcinoma antigen (SCCA), fail to show any discrimination. Additionally, combining the six-protein panel with SCCA improves the discrimination of patients with CES and CLS from healthy controls.

HPLC analysis of related impurities contents in Drotaverine hydrochloride substances was done by using various methods of quality control in accordance with the requirements of substances and drugs manufacturers. It is shown, that different chromatographic techniques give different results in the control of the same series of Drotaverine hydrochloride substance. Comparative study of the relationships between abnormal toxicity and total impurity contents are not allowed to chose an acceptable HPLC method for related impurities control in the investigated substances.

Proteome profiling of crude serum is a challenging task due to the wide dynamic range of protein concentrations and the presence of high-abundance proteins, which cover >90% of the total protein mass in serum. Peptide fractionation on strong cation exchange, weak anion exchange in the electrostatic repulsion hydrophilic interaction chromatography (ERLIC) mode, RP C18 at pH 2.5 (low pH), fused-core fluorinated at pH 2.5, and RP C18 at pH 9.7 (high pH) stationary phases resulted in two to three times more identified proteins and three to four times more identified peptides in comparison with 1D nanoChip-LC-MS/MS quadrupole TOF analysis (45 proteins, 185 peptides). The largest number of peptides and proteins was identified after prefractionation in the ERLIC mode due to the more uniform distribution of peptides among the collected fractions and on the RP column at high pH due to the high efficiency of RP separations and the complementary selectivity of both techniques to low-pH RP chromatography. A 3D separation scheme combining ERLIC, high-pH RP, and low-pH nanoChip-LC-MS/MS for crude serum proteome profiling resulted in the identification of 208 proteins and 1088 peptides with the lowest reported concentration of 11 ng/mL for heat shock protein 74.