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.

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.

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.

Development and validation of ultra-high performance supercritical fluid chromatography method for quantitative determination of nine sunscreens in cosmetic samples.

A fast and simple utra-high performance supercritical fluid chromatography (UHPSFC) method has been developed for the determination of nine sunscreens (UV filters), namely 2-ethylhexyl-2-hydroxybenzoate (ES), ethylhexyl-methoxycinnamate (EMC), benzophenone-3 (BZ3), octocrylene (OCR), bis-ethylhexyloxyphenol-methoxyphenyl triazine (EMT), butyl-methoxydibenzoyl-methane (BDM), diethylamino-hydroxybenzoyl-hexyl-benzoate (DHB), ethylhexyl-triazone (ET), and diethylhexyl-butamido-triazone (DBT) in cosmetic samples. The separation was achieved with Acquity UPC2 Torus 2-PIC (100 × 3.0 mm, 1.7 µm) column. The influence of key chromatographic parameters on resolution was evaluated. The optimal mobile phase was a gradient mixture of carbon dioxide and methanol containing ammonium acetate, at flow rate 1.2 mL min-1. The back-pressure of the system was set to 150 bar and the temperature to 40 °C. The compounds were determined with diode-array detection at 280 nm, 305 nm and 340 nm depending on absorbance maxima. The proposed UHPSFC method provided separation of the nine target sunscreens within 2.5 min without labor-consuming sample pretreatment procedure. The method was validated according to the concept of the total error and the accuracy profile. The inter-laboratory reproducibility was evaluated between two independent laboratories (in France and Czech Republic). The reliability of the developed method was shown by application to commercial sunscreen-containing cosmetic sample obtained from French market. The measured limits of quantification showed the suitability of the proposed method for determination of UV filters considering the European Union requirements.

Sum of ranking differences to rank stationary phases used in packed column supercritical fluid chromatography.

The identification of a suitable stationary phase in supercritical fluid chromatography (SFC) is a major source of difficulty for those with little experience in this technique. Several protocols have been suggested for column classification in high-performance liquid chromatography (HPLC), gas chromatography (GC), and SFC. However, none of the proposed classification schemes received general acceptance. A fair way to compare columns was proposed with the sum of ranking differences (SRD). In this project, we used the retention data obtained for 86 test compounds with varied polarity and structure, analyzed on 71 different stationary phases encompassing the full range in polarity of commercial packed columns currently available to the SFC chromatographer, with a single set of mobile phase and operating conditions (carbon dioxide-methanol mobile phase, 25°C, 150bar outlet pressure, 3ml/min). First, a reference column was selected and the 70 remaining columns were ranked based on this reference column and the retention data obtained on the 86 analytes. As these analytes previously served for the calculation of linear solvation energy relationships (LSER) on the 71 columns, SRD ranks were compared to LSER methodology. Finally, an external comparison based on the analysis of 10 other analytes (UV filters) related the observed selectivity to SRD ranking. Comparison of elution orders of the UV filters to the SRD rankings is highly supportive of the adequacy of SRD methodology to select similar and dissimilar columns.

Development and validation of ultra-high performance supercritical fluid chromatography method for determination of illegal dyes and comparison to ultra-high performance liquid chromatography method.

A novel simple, fast and efficient ultra-high performance supercritical fluid chromatography (UHPSFC) method was developed and validated for the separation and quantitative determination of eleven illegal dyes in chili-containing spices. The method involved a simple ultrasound-assisted liquid extraction of illegal compounds with tetrahydrofuran. The separation was performed using a supercritical fluid chromatography system and CSH Fluoro-Phenyl stationary phase at 70°C. The mobile phase was carbon dioxide and the mixture of methanol:acetonitrile (1:1, v/v) with 2.5% formic acid as an additive at the flow rate 2.0 mL min(-1). The UV-vis detection was accomplished at 500 nm for seven compounds and at 420 nm for Sudan Orange G, Butter Yellow, Fast Garnet GBC and Methyl Red due to their maximum of absorbance. All eleven compounds were separated in less than 5 min. The method was successfully validated and applied using three commercial samples of chili-containing spices - Chili sauce (Indonesia), Feferony sauce (Slovakia) and Mojo sauce (Spain). The linearity range of proposed method was 0.50-9.09 mg kg(-1) (r ≥ 0.995). The detection limits were determined as signal to noise ratio of 3 and were ranged from 0.15 mg kg(-1) to 0.60 mg kg(-1) (1.80 mg kg(-1) for Fast Garnet) for standard solution and from 0.25 mg kg(-1) to 1.00 mg kg(-1) (2.50 mg kg(-1) for Fast Garnet, 1.50 mg kg(-1) for Sudan Red 7B) for chili-containing samples. The recovery values were in the range of 73.5-107.2% and relative standard deviation ranging from 0.1% to 8.2% for within-day precision and from 0.5% to 8.8% for between-day precision. The method showed potential for being used to monitor forbidden dyes in food constituents. The developed UHPSFC method was compared to the UHPLC-UV method. The orthogonality of Sudan dyes separation by these two methods was demonstrated. Benefits and drawbacks were discussed showing the reliability of both methods for monitoring of studied illegal dyes in real food constituents.

On-line SPE-UHPLC method using fused core columns for extraction and separation of nine illegal dyes in chilli-containing spices.

The presented work describes the development of a simple, fast and effective on-line SPE-UHPLC-UV/vis method using fused core particle columns for extraction, separation and quantitative analysis of the nine illegal dyes, most frequently found in chilli-containing spices. The red dyes Sudan I-IV, Sudan Red 7B, Sudan Red G, Sudan Orange G, Para Red, and Methyl Red were separated and analyzed in less than 9 min without labor-consuming pretreatment procedure. The chromatographic separation was performed on Ascentis Express RP-Amide column with gradient elution using mixture of acetonitrile and water, as a mobile phase at a flow rate of 1.0 mL min(-1) and 55°C of temperature. As SPE sorbent for cleanup and pre-concentration of illegal dyes short guard fused core column Ascentis Express F5 was used. The applicability of proposed method was proven for three different chilli-containing commercial samples. Recoveries for all compounds were between 90% and 108% and relative standard deviation ranged from 1% to 4% for within- and from 2% to 6% for between-day. Limits of detection showed lower values than required by European Union regulations and were in the range of 3.3-10.3 µg L(-1) for standard solutions, 5.6-235.6 µg kg(-1) for chilli-containing spices.