Mitigating In-Column Artificial Modifications in High-Temperature LC-MS for Bottom-Up Proteomics and Quality Control of Protein Biopharmaceuticals.

Elevating the column temperature is an effective strategy for improving the chromatographic separation of peptides. However, high temperatures induce artificial modifications that compromise the quality of the peptide analysis. Here, we present a novel high-temperature LC-MS method that retains the benefits of a high column temperature while significantly reducing peptide modification and degradation during reversed-phase liquid chromatography. Our approach leverages a short inline trap column maintained at a near-ambient temperature installed upstream of a separation column. The retentivity and dimensions of the trap column were optimized to shorten the residence time of peptides in the heated separation column without compromising the separation performance. This easy-to-implement approach increased peak capacity by 1.4-fold within a 110 min peptide mapping of trastuzumab and provided 10% more peptide identifications in exploratory LC-MS proteomic analyses compared with analyses conducted at 30 °C while maintaining the extent of modifications close to the background level. In the peptide mapping of biopharmaceuticals, where in-column modifications can falsely elevate the levels of some critical quality attributes, the method reduced temperature-related artifacts by 66% for N-terminal pyroGlu and 63% for oxidized Met compared to direct injection at 60 °C, thus improving reliability in quality control of protein drugs. Our findings represent a promising advancement in LC-MS methodology, providing researchers and industry professionals with a valuable tool for improving the chromatographic separation of peptides while significantly reducing the unwanted modifications.

Easy, Robust, and Repeatable Online Acid Cleavage of Proteins in Mobile Phase for Fast Quantitative LC-MS Bottom-Up Protein Analysis─Application for Ricin Detection.

Sample preparation involving the cleavage of proteins into peptides is the first critical step for successful bottom-up proteomics and protein analyses. Time- and labor-intensiveness are among the bottlenecks of the commonly used methods for protein sample preparation. Here, we report a fast online method for postinjection acid cleavage of proteins directly in the mobile phase typically used for LC-MS analyses in proteomics. The chemical cleavage is achieved in 0.1% formic acid within 35 s in a capillary heated to 195 °C installed upstream of the analytical column, enabling the generated peptides to be separated. The peptides generated by the optimized method covered the entire sequence except for one amino acid of trastuzumab used for the method development. The qualitative results are extraordinarily stable, even over a long period of time. Moreover, the method is also suitable for accurate and repeatable quantification. The procedure requires only one manual step, significantly decreasing sample transfer losses. To demonstrate its practical utility, we tested the method for the fast detection of ricin. Ricin can be unambiguously identified from an injection of 10 ng, and the results can be obtained within 7-8 min after receiving a suspicious sample. Because no sophisticated accessories and no additional reagents are needed, the method can be seamlessly transferred to any laboratory for high-throughput proteomic workflows.

A stationary phase with a positively charged surface allows for minimizing formic acid concentration in the mobile phase, enhancing electrospray ionization in LC-MS proteomic experiments.

The default choice of mobile phase acidifier for bottom-up LC-MS proteomic analyses is 0.10% formic acid because of its decent acidity, decent ion pairing ability, and low suppression of electrospray ionization. In recent years, state-of-the-art columns have been designed specifically to provide efficient separation even when using an MS-friendly mobile phase of low ionic strength. Despite this, no attempts have been made to improve the sensitivity of the MS-based analytical methods by reducing the amount of formic acid in the mobile phase. In this study, we evaluated the effect of reduced formic acid concentration in the mobile phase on the chromatographic behavior and MS response of peptides when separated using columns packed with a C18 stationary phase with a positively charged surface. Using 0.01% formic acid in the mobile phase maintained excellent chromatographic performance and increased MS signal response compared to the standard of 0.10%. The enhanced MS response translated to about 50% improved peptide identifications depending on the complexity and amount of sample injected. The increased retention of peptides at a reduced formic acid concentration was directly proportional to the number of acidic residues in the peptide sequence. The study was carried out by covering a spectrum of protein samples with varied complexity using analytical flow, micro-, and nanoflow regimes to expand the applicability in routine practice.

Reversed-Phase Liquid Chromatography of Peptides for Bottom-Up Proteomics: A Tutorial.

The performance of the current bottom-up liquid chromatography hyphenated with mass spectrometry (LC-MS) analyses has undoubtedly been fueled by spectacular progress in mass spectrometry. It is thus not surprising that the MS instrument attracts the most attention during LC-MS method development, whereas optimizing conditions for peptide separation using reversed-phase liquid chromatography (RPLC) remains somewhat in its shadow. Consequently, the wisdom of the fundaments of chromatography is slowly vanishing from some laboratories. However, the full potential of advanced MS instruments cannot be achieved without highly efficient RPLC. This is impossible to attain without understanding fundamental processes in the chromatographic system and the properties of peptides important for their chromatographic behavior. We wrote this tutorial intending to give practitioners an overview of critical aspects of peptide separation using RPLC to facilitate setting the LC parameters so that they can leverage the full capabilities of their MS instruments. After briefly introducing the gradient separation of peptides, we discuss their properties that affect the quality of LC-MS chromatograms the most. Next, we address the in-column and extra-column broadening. The last section is devoted to key parameters of LC-MS methods. We also extracted trends in practice from recent bottom-up proteomics studies and correlated them with the current knowledge on peptide RPLC separation.

Sense and Nonsense of Elevated Column Temperature in Proteomic Bottom-up LC-MS Analyses.

Elevated column temperature represents a simple means for improving chromatographic separation of peptides. Here, we demonstrated the advantages of the column temperature in peptide separation using state-of-the-art columns. More importantly, we also determined how temperature can impair proteomic bottom-up analyses. We found that an elevated temperature in combination with the acidic pH of the mobile phase induced in-column peptide hydrolysis with high specificity to Asp and accelerated five modification reactions of amino acids. The positive effects of temperature dominated in the 30 min long gradients since the column operated at 90 °C provided the largest number of identified peptides and proteins. However, the adverse effects of temperature on peptide integrity in longer liquid chromatography-mass spectrometry (LC-MS) analyses required its reduction to obtain optimum results. The largest number of peptides was identified using the column maintained at 75 °C in 60 min long gradients, at 60 °C in 120 min long gradients, and at 45 °C in 240 min long gradients. Our results indicate that no universal column temperature exists for bottom-up LC-MS analyses. Quite the contrary, the temperature setting must be selected rationally to exploit the full capabilities of the state-of-the-art mass spectrometers in proteomic LC-MS analyses, with the gradient time being a critical factor.

Dissolving Peptides in 0.1% Formic Acid Brings Risk of Artificial Formylation.

The ability of concentrated formic acid to formylate reactive amino acid residues is known from previous reports. In contrast, solvents containing a low concentration of formic acid are generally recognized as a safe environment for proteomic applications. The primary objective of this study was to explain the excessive formylation rate in tryptic peptides that did not come into contact with concentrated formic acid. We found out that the peptide formylation was associated with dissolving the peptides in a solvent containing mere 0.1% formic acid. Similar conclusions were drawn after analyzing publicly available proteomic data. We further demonstrated that these unwanted modifications can be averted via handling the samples at a low temperature or, obviously, via replacing formic acid in the solvent with trifluoroacetic acid. These simple countermeasures can contribute to a reduction in the part of the MS/MS spectra that remain unassigned to a peptide sequence.

Conventional-Flow Liquid Chromatography-Mass Spectrometry for Exploratory Bottom-Up Proteomic Analyses.

Due to its sensitivity and productivity, bottom-up proteomics based on liquid chromatography-mass spectrometry (LC-MS) has become the core approach in the field. The de facto standard LC-MS platform for proteomics operates at sub-µL/min flow rates, and nanospray is required for efficiently introducing peptides into a mass spectrometer. Although this is almost a "dogma", this view is being reconsidered in light of developments in highly efficient chromatographic columns, and especially with the introduction of exceptionally sensitive MS instruments. Although conventional-flow LC-MS platforms have recently penetrated targeted proteomics successfully, their possibilities in discovery-oriented proteomics have not yet been thoroughly explored. Our objective was to determine what are the extra costs and what optimization and adjustments to a conventional-flow LC-MS system must be undertaken to identify a comparable number of proteins as can be identified on a nanoLC-MS system. We demonstrate that the amount of a complex tryptic digest needed for comparable proteome coverage can be roughly 5-fold greater, providing the column dimensions are properly chosen, extra-column peak dispersion is minimized, column temperature and flow rate are set to levels appropriate for peptide separation, and the composition of mobile phases is fine-tuned. Indeed, we identified 2 835 proteins from 2 µg of HeLa cells tryptic digest separated during a 60 min gradient at 68 µL/min on a 1.0 mm × 250 mm column held at 55 °C and using an aqua-acetonitrile mobile phases containing 0.1% formic acid, 0.4% acetic acid, and 3% dimethyl sulfoxide. Our results document that conventional-flow LC-MS is an attractive alternative for bottom-up exploratory proteomics.

Targeted proteomics driven verification of biomarker candidates associated with breast cancer aggressiveness.

Breast cancer is the most common and molecularly relatively well characterized malignant disease in women, however, its progression to metastatic cancer remains lethal for 78% of patients 5years after diagnosis. Novel markers could identify the high risk patients and their verification using quantitative methods is essential to overcome genetic, inter-tumor and intra-tumor variability and translate novel findings into cancer diagnosis and treatment. We recently identified 13 proteins associated with estrogen receptor, tumor grade and lymph node status, the key factors of breast cancer aggressiveness, using untargeted proteomics. Here we verified these findings in the same set of 96 tumors using targeted proteomics based on selected reaction monitoring with mTRAQ labeling (mTRAQ-SRM), transcriptomics and immunohistochemistry and validated in 5 independent sets of 715 patients using transcriptomics. We confirmed: (i) positive association of anterior gradient protein 2 homolog (AGR2) and periostin (POSTN) and negative association of annexin A1 (ANXA1) with estrogen receptor status; (ii) positive association of stathmin (STMN1), cofilin-1 (COF1), plasminogen activator inhibitor 1 RNA-binding protein (PAIRBP1) and negative associations of thrombospondin-2 (TSP2) and POSTN levels with tumor grade; and (iii) positive association of POSTN, alpha-actinin-4 (ACTN4) and STMN1 with lymph node status. This study highlights a panel of gene products that can contribute to breast cancer aggressiveness and metastasis, the understanding of which is important for development of more precise breast cancer treatment.

Proteomic investigation of embryonic rat heart-derived H9c2 cell line sheds new light on the molecular phenotype of the popular cell model.

Due to their cardiac origin, H9c2 cells rank among the most popular cell lines in current cardiovascular research, yet molecular phenotype remains elusive. Hence, in this study we used proteomic approach to describe molecular phenotype of H9c2 cells in their undifferentiated (i.e., most frequently used) state, and its functional response to cardiotoxic drug doxorubicin. Of 1671 proteins identified by iTRAQ IEF/LC-MSMS analysis, only 12 proteins were characteristic for striated muscle cells and none was cardiac phenotype-specific. Targeted LC-SRM and western blot analyses confirmed that undifferentiated H9c2 cells are phenotypically considerably different to both primary neonatal cardiomyocytes and adult myocardium. These cells lack proteins essential for formation of striated muscle myofibrils or they express only minor amounts thereof. They also fail to express many proteins important for metabolism of muscle cells. The challenge with clinically relevant concentrations of doxorubicin did not induce a proteomic signature that has been previously noted in primary cardiomyocytes or adult hearts. Instead, several alterations previously described in other cells of mesodermal origin, such as fibroblasts, were observed (e.g., severe down-regulation of collagen synthesis pathway). In conclusion, the molecular phenotype of H9c2 cells resembles very immature myogenic cells with skeletal muscle commitment upon differentiation and thus, translatability of findings obtained in these cells deserves caution.

Changes in proteome of the Δhfq strain derived from Francisella tularensis LVS correspond with its attenuated phenotype.

The posttranscriptional regulatory protein Hfq was shown to be an important determinant of the stress resistance and full virulence in the dangerous human pathogen Francisella tularensis. Transcriptomics brought rather limited clues to the precise contribution of Hfq in virulence. To reveal the molecular basis of the attenuation caused by hfq inactivation, we employed iTRAQ in the present study and compared proteomes of the parent and isogenic Δhfq strains. We show that Hfq modulates the level of 76 proteins. Most of them show decreased abundance in the ∆hfq mutant, thereby indicating that Hfq widely acts rather as a positive regulator of Francisella gene expression. Several key Francisella virulence factors including those encoded within the Francisella pathogenicity island were found among the downregulated proteins, which is in a good agreement with the attenuated phenotype of the Δhfq strain. To further validate the iTRAQ exploratory findings, we subsequently performed targeted LC-SRM analysis of selected proteins. This accurate quantification method corroborated the trends found in the iTRAQ data.

Role of steric hindrance in the Newman-Kwart rearrangement and in the synthesis and photophysical properties of arylsulfanyl tetrapyrazinoporphyrazines.

Conditions for the Newman-Kwart rearrangement of phenols into thiophenols were investigated in relation to the bulkiness of substituents at the 2 and 6 positions of the starting phenol derivative with an emphasis on eliminating side reactions. Thiophenols with different 2,6-disubstitution patterns (including hydrogen, methyl, isopropyl or tert-butyl groups) were used for the synthesis of 5,6-bis(arylsulfanyl)pyrazine-2,3-dicarbonitriles that underwent cyclotetramerization leading to the corresponding zinc tetrapyrazinoporphyrazines (TPyzPz), aza-analogues of phthalocyanines. Several methods for the cyclotetramerization were attempted to eliminate problematic side reactions. Magnesium butoxide was found as the most suitable cyclotetramerization agent and afforded TPyzPzs in reasonable yields of approximately 30% under mild conditions. The varying arrangements of the peripheral substitutions resulting from the different bulkiness of the substituents were demonstrated by the X-ray structures of the pyrazine-2,3-dicarbonitriles. The prepared zinc arylsulfanyl TPyzPzs showed an absorption maximum at a Q-band over 650 nm, fluorescence quantum yields between 0.078 and 0.20, and singlet oxygen quantum yields ranging 0.58-0.69. TPyzPzs with isopropyl groups were found to be the best derivatives in this series as they combined facile cyclotetramerization, no aggregation, and good photophysical properties, which makes them potentially suitable for photodynamic therapy.

Structural factors influencing the intramolecular charge transfer and photoinduced electron transfer in tetrapyrazinoporphyrazines.

A series of unsymmetrical tetrapyrazinoporphyrazines (TPyzPzs) from the group of azaphthalocyanines with one peripherally attached amino substituent (donor) were synthesized, and their photophysical properties (fluorescence quantum yield and singlet oxygen quantum yield) were determined. The synthesized TPyzPzs were expected to undergo intramolecular charge transfer (ICT) as the main pathway for deactivating their excited states. Several structural factors were found to play a critical role in ICT efficiency. The substituent in the ortho position to the donor center significantly influences the ICT, with tert-butylsulfanyl and butoxy substituents inducing the strongest ICTs, whereas chloro, methyl, phenyl, and hydrogen substituents in this position reduce the efficiency. The strength of the donor positively influences the ICT efficiency and correlates well with the oxidation potential of the amines used as the substituents on the TPyzPz as follows: n-butylamine < N,N-diethylamine < aniline < phenothiazine. The ICT (with conjugated donors and acceptors) in the TPyzPz also proved to be much stronger than a photoinduced electron transfer in which the donor and the acceptor are connected through an aliphatic linker.

Detection of novel auto-antigens in patients with recurrent miscarriage: description of an approach and preliminary findings.

AIM: To develop and test a protocol for isolation of potential auto-antigens from chorionic tissue that may be linked to recurrent miscarriage (RM). METHODS: The strategy included: 1) isolation of IgGs tightly bound to chorionic tissue of RM patients by protein G chromatography; 2) construction of affinity columns using the chorionic antibodies for isolation of auto-antigens; 3) enrichment of auto-antigens from detergent extracted solution of chorionic proteins by affinity chromatography; 4) separation by dodecyl sulfate-electrophoresis followed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry identification. RESULTS: Five potential auto-antigens were detected: neutral alpha-glucosidase AB, endoplasmin, transitional endoplasmic reticulum ATPase, putative endoplasmin-like protein, and cytoplasmic actin 2. CONCLUSIONS: We developed a strategy for identification of auto-antigens in the chorionic tissue of women with RM, which could be of diagnostic and prognostic value.

Evaluating C18 stationary phases with a positively charged surface for proteomic LC-MS applications using mobile phase acidified with reduced formic acid concentration.

We have recently demonstrated the ability of a C18 stationary phase with a positively charged surface (PCS-C18) to provide superior chromatographic separation of peptides using mobile phase acidified with a mere 0.01 % formic acid, significantly improving MS sensitivity. Here, we examined three columns packed with different PCS-C18 phases using the MS-favorable mobile phase acidified with low formic acid concentrations to establish the impact of separation performance and better MS sensitivity on peptide identifications. The surface charge interaction was evaluated using the retention of nitrate. The highest interaction was observed for the AdvanceBio Peptide Plus column. A surface charge-dependent shift in the retention time of peptides was confirmed with a change in formic acid concentration in the mobile phase. The separation performance of the columns with MS-favorable mobile phase acidified with low concentrations of formic acid was evaluated using well-characterized peptides. The loading capacity was assessed using a basic peptide with three lysine residues. Good chromatographic peak shapes and high loading capacity were observed for the Acquity Premier CSH C18 column, even when using a mobile phase acidified with 0.01 % formic acid. The extent of improvement in peptide identification when using reduced formic acid concentration was evaluated by analyzing the tryptic digest of trastuzumab and tryptic digest of whole bacteria cell lysate. Each column provided improved MS signal intensity and peptide identification when using the mobile phase with 0.01 % formic acid. The ability of the Acquity Premier CSH C18 column to provide better separation of peptides, even with a reduced formic acid concentration in the mobile phase, boosted MS signal intensity by 65 % and increased the number of identified peptides from the bacterial sample by 19 %. Our study confirms that significant improvement in the proteomic outputs can be achieved without additional costs only by tailoring the chemistry of the stationary phase to the composition of the mobile phase. Our results can help researchers understand the retention mechanism of peptides on the PCS-C18 stationary phases using low-ionic strength mobile phases and, more importantly, select the best-suited stationary phases for their LC-MS proteomic applications.

Implementing reversed-phase and hydrophilic interaction liquid chromatography into the characterization of DTPA-ramucirumab conjugate before radiolabeling.

Radioimmunoconjugates represent a promising class of therapeutics and diagnostics. The characterization of intermediate chelator-antibody products, i.e., without the radionuclide, is frequently omitted, bringing significant uncertainty in the radioimmunoconjugate preparation. In the present study, we explored the utility of reversed-phase (RPLC) and hydrophilic interaction (HILIC) liquid chromatography with UV detection to characterize ramucirumab stochastically conjugated with p-SCN-Bn-CHX-A"-DTPA chelator (shortly DTPA). The conjugation was well reflected in RPLC chromatograms, while chromatograms from HILIC were significantly less informative. RPLC analyses at the intact level confirmed that the conjugation resulted in a heterogeneous mixture of modified ramucirumab. Moreover, the RPLC of DTPA-ramucirumab confirmed heterogeneous conjugation of all subunits. The peptide mapping did not reveal substantial changes after the conjugation, indicating that most parts of ramucirumab molecules remained unmodified and that the DTPA chelator was bound to various sites. Eventually, the RPLC method for analysis of intact ramucirumab was successfully applied to online monitoring of conjugation reaction in 1 h intervals for a total of 24 h synthesis, which readily reflected the structural changes of ramucirumab in the form of retention time shift by 0.21 min and increase in peak width by 0.22 min. The results were obtained in real-time, practically under 10 min per monitoring cycle. To the best of our knowledge, our study represents the first evaluation of RPLC and HILIC to assess the quality of intermediates during the on-site preparation of radioimmunoconjugates prior to radiolabeling.

MS/MS library facilitated MRM quantification of native peptides prepared by denaturing ultrafiltration.

Naturally occurring native peptides provide important information about physiological states of an organism and its changes in disease conditions but protocols and methods for assessing their abundance are not well-developed. In this paper, we describe a simple procedure for the quantification of non-tryptic peptides in body fluids. The workflow includes an enrichment step followed by two-dimensional fractionation of native peptides and MS/MS data management facilitating the design and validation of LC- MRM MS assays. The added value of the workflow is demonstrated in the development of a triplex LC-MRM MS assay used for quantification of peptides potentially associated with the progression of liver disease to hepatocellular carcinoma.

Proteomic insights into chronic anthracycline cardiotoxicity.

Chronic anthracycline cardiotoxicity is a feared complication of cancer chemotherapy. However, despite several decades of primarily hypothesis-driven research, the molecular basis of this phenomenon remains poorly understood. The aim of this study was to obtain integrative molecular insights into chronic anthracycline cardiotoxicity and the resulting heart failure. Cardiotoxicity was induced in rabbits (daunorubicin 3mg/kg, weekly, 10weeks) and changes in the left ventricular proteome were analyzed by 2D-DIGE. The protein spots with significant changes (p<0.01, >1.5-fold) were identified using MALDI-TOF/TOF. Key data were corroborated by immunohistochemistry, qRT-PCR and enzyme activity determination and compared with functional, morphological and biochemical data. The most important alterations were found in mitochondria - especially in proteins crucial for oxidative phosphorylation, energy channeling, antioxidant defense and mitochondrial stress. Furthermore, the intermediate filament desmin, which interacts with mitochondria, was determined to be distinctly up-regulated and disorganized in its expression pattern. Interestingly, the latter changes reflected the intensity of toxic damage in whole hearts as well as in individual cells. In addition, a marked drop in myosin light chain isoforms, activation of proteolytic machinery (including the proteasome system), increased abundance of chaperones and proteins involved in chaperone-mediated autophagy, membrane repair as well as apoptosis were found. In addition, dramatic changes in proteins of basement membrane and extracellular matrix were documented. In conclusion, for the first time, the complex proteomic signature of chronic anthracycline cardiotoxicity was revealed which enhances our understanding of the basis for this phenomenon and it may enhance efforts in targeting its reduction.

Evaluation of strategies for overcoming trifluoroacetic acid ionization suppression resulted in single-column intact level, middle-up, and bottom-up reversed-phase LC-MS analyses of antibody biopharmaceuticals.

A wide range of strategies for efficient chromatography and high MS sensitivity in reversed-phase LC-MS analysis of antibody biopharmaceuticals and their large derivates has been evaluated. They included replacing trifluoroacetic acid with alternative acidifiers, relevancy of elevated column temperature, use of dedicated stationary phases, and counteraction of the suppression effect of trifluoroacetic acid in electrospray ionization. At the column temperature of 60 °C, which significantly reduces in-column protein degradation, the BioResolve RP mAb Polyphenyl, BioShell IgG C4 columns performed best using mobile phases with full or partial replacement of trifluoroacetic acid with difluoroacetic acid in the analysis of intact antibodies. Similarly, 0.03% trifluoroacetic acid in combination with 0.07% formic acid is a good alternative in analyzing antibody chains at 60 °C. Collectively, the addition of 3% 1-butanol to the mobile phase acidified with 0.1% formic acid was the most efficient approach to simultaneously achieving good chromatographic separation and MS sensitivity for intact and reduced antibody biopharmaceuticals. Moreover, this mobile phase combined with the BioResolve RP mAb Polyphenyl column was subsequently demonstrated to provide excellent results for peptide mapping of antibody biopharmaceuticals fully comparable with those obtained using a state-of-the-art column for peptide separation, thus opening an avenue for a single-column multilevel analysis of these biotherapeutics.

Comparative proteomic profiling of culture filtrate proteins of less and highly virulent Francisella tularensis strains.

The facultative intracellular bacterium Francisella tularensis is the causal agent of the serious infectious disease tularemia. Despite the dynamic progress, which has been made in last few years, important questions regarding Francisella pathogenicity still remain to be answered. Generally, secreted proteins play an important role in pathogenicity of intracellular microbes. In this study, we investigated the protein composition of the culture filtrate proteins of highly virulent F. tularensis subsp. tularensis, strain SCHU S4 and attenuated F. tularensis subsp. holarctica, live vaccine strain using a comparative proteomic analysis. The majority of proteins identified in this study have been implicated in virulence mechanisms of other pathogens, and several have been categorized as having moonlighting properties; those that have more than one unrelated function. This profiling study of secreted proteins resulted in the unique detection of acid phosphatase (precursor) A (AcpA), ß-lactamase, and hypothetical protein FTT0484 in the highly virulent strain SCHU S4 secretome. The release of AcpA may be of importance for F. tularensis subsp. tularensis virulence due to the recently described AcpA role in the F. tularensis escape from phagosomes.