Application Note LCMS-112 A Fully Automated Two-Step Procedure for Quality Control of Synthetic Peptides

Transcription

1 Application Note LCMS-112 A Fully Automated Two-Step Procedure for Quality Control of Synthetic Peptides Abstract Here we describe a two-step QC procedure for synthetic peptides. In the first step, the identity of the peptide is confirmed by comparing theoretical and measured mass. In a second step, the purity of the peptide is calculated from the peak areas in the base peak chromatogram. The fully automated procedure provides easily interpretable results that provide at-a-glance conformation of the presence and purity of the target peptide. Introduction Mass spectrometry is the preferred analytical technique used for the quality control of synthetic peptides. It can provide the molecular mass of peptides and also fragment information that can help confirm the identity of the expected product. When used in combination with reversed-phase chromatography, it is the most common technique for the determination of the purity of peptides [1]. Authors Maik Henkel Forschungszentrum Borstel (FBZ), Leibniz-Zentrum für Medizin und Biowissenschaften, Germany Zoltan Czentnar, Andrea Kiehne Bruker Daltonik GmbH, Bremen, Germany Keywords Instrumentation and Software Peptide QC amazon SL Ion Trap Compass OpenAccess 1.4 Automation Peptides are usually synthesized on a solid support. This methodology was first described by Bruce Merrifield in 1963 [2] and enables rapid synthesis of large numbers of peptides in an automated process.

2 The peptides are synthesized by stepwise elongation of a peptide chain using amino acid building blocks. Because peptides are biopolymers consisting of amino acids with known masses, the expected molecular weight of the synthesized peptide is relatively easy to calculate from the respective amino acid building blocks. Typically, peptides are synthesized by automated highthroughput peptide synthesizers, either in parallel reaction vessels for medium-scale syntheses or in 96-well plates for small-scale syntheses. These methods enable the synthesis of hundreds of peptides in a single batch. Due to the large number of synthesis products, manual data acquisition and processing for quality-control purposes is inefficient. Moreover, manually calculating the molecular mass of every peptide in the synthesis is very time-consuming and prone to errors, especially when peptides contain non-standard amino acids or special modifications. For this reason, we developed an automated procedure for the quality control of synthesized peptides using the Bruker Compass OpenAccess QC software package. The established, easy-to-use workflow enables efficient and accurate quality control of the synthesis products. The user only needs to submit a sample table with a sample name and expected peptide sequence, choose the desired pre-defined LC-MS method, and place the samples in the autosampler of the HPLC system. The peptides are automatically analyzed and the result reports provide all necessary information on the presence and purity of the expected peptides (Fig 1). Peptide QC workflow Figure 1: Peptide QC workflow from sample injection to result report.

3 Experimental Instrumentation The LC-MS system consists of an amazon SL ion trap mass spectrometer (Bruker Daltonics) with a standard Apollo II ESI source and an UltiMate 3000 RSLC system (Thermo Fisher Scientific). The setup was equipped with the Compass OpenAccess software package for automated routine peptide QC analysis. The following source parameters were applied for operation in positive ESI mode: capillary voltage 4000 V, nebulizer gas 25 psi, dry gas 8 L/min, dry temperature 320 C. Peptide verification method First, the identity of the peptides from the synthesis laboratory is verified. Sample information and peptide sequences are submitted to Compass OpenAccess using an Excel csv file. The samples are placed in the HPLC autosampler and the Peptide Verification method is started using Compass OpenAccess. After loop injection, full-scan MS spectra are recorded in positive ion mode for 2 minutes to determine the peptide molecular mass. Using this short method, a throughput of raw peptide samples per day can be achieved. If the expected peptide is detected and the first QC test is passed, the sample can be used directly. For applications which require a specific purity, the peptide also needs to be analyzed with the purity method. Table 1: HPLC Conditions LC Settings HPLC system Eluent A Eluent B Injection volume 2 µl Peptide Verification Method Loop injection Isocratic elution Flow rate Peptide Purity Method Column UltiMate 3000 RSLC 0.1% Formic acid in water 0.1% Formic acid in acetonitrile Eluent A/B (45/55 ; v/v) 0.3 ml/min Phenomenex, Aeris PEPTIDE 1.7 µm XB-C mm Gradient 0.0 min; 15% B Flow rate 13.0 min; 45% B 13.1 min; 90% B 14.0 min; 90% B 14.1 min; 15% B 15.0 min; 15% B 0.4 ml/min Peptide purity method In the second step of the peptide QC workflow, the purity of the peptides is determined. For this purpose, all samples of interest are subjected to Peptide Purity analysis using Compass OpenAccess. In this analysis, samples are separated using a 15 min HPLC method resulting in a theoretical throughput of samples per day. Full-scan MS Spectra are recorded in positive ion mode. Details of HPLC and MS methods can be found in Tables 1 and 2. Data processing Data processing (including reporting) is fully automatic. Masses of amino acid building blocks and modifications are stored in Excel spread sheets and are used to calculate the monoisotopic theoretical masses of the synthesized peptides. Amino acid and modification tables can be edited by the user to add new or delete obsolete entries. Table 2: MS Parameters (SPS = Smart Parameter Setting) MS Parameters Ionization Polarity Tuning Ion Charge Control Target Electrospray (ESI) Positive ion mode SPS for target mass 700 m/z Scan mode Enhanced (8100 m/z sec -1 ) Scan range m/z

4 Results For the verification of peptide identity, an extracted ion chromatogram (EIC) is generated based on the sum of the expected m/z values of charge states 2 to 6. The acquired full-scan MS data is averaged between 0.3 and 1.2 minutes and the resulting spectrum is deconvoluted to determine the neutral peptide mass. The main signal in the deconvoluted spectrum is compared to the calculated theoretical peptide molecular weight. If the deviation of the experimental monoisotopic mass is less than ±0.5 Da, the peptide identity is considered to be correct. The results of the peptide verification are provided in a PDF report that is stored on the Compass OpenAccess Server and the acquisition computer. A typical report is shown in Fig. 2. Base peak chromatogram (BPC) and EIC are displayed in the upper part of the report. The lower part displays the expected sequence and the corresponding monoisotopic neutral peptide mass. In this case, the sequence was Q P M A Nle V Q S V P Q, where Nle is the uncommon amino acid norleucine and an example for the simple incorporation of special amino acids in the corresponding building block used by OpenAccess. As the final result, the report contains the information Expected peptide found. The measured full scan MS spectrum and the corresponding deconvoluted spectrum in the lower half of the report show the details of the experimental results. In a second step, the synthesis products are submitted to the peptide purity check. In this workflow, peak areas of the target peptide and impurities are determined after HPLC separation. The peptide purity is calculated by dividing the peak area of the peptide by the total peak area (determined by integration of all peaks present in the base peak chromatogram). From this ratio, the relative peptide percentage purity is derived. In addition to the purity calculation, the mass of the target peptide is confirmed once again using the same procedure as for the peptide identity verification. Peptide verification report with sample ID Figure 2: Peptide verification report with sample ID, peptide sequence and expected monoisotopic molecular weight ( Da). The experimental mass Mr = Da is given in the deconvoluted spectrum. Here, the mass error Δm = 0.01 Da is well within the threshold of ±0.5 Da.

5 Fig. 3 shows a PDF report from peptide purity determination. The upper panel displays the base chromatogram with integration of the major peak (no. 2) and a smaller peak (no. 1). The extracted ion chromatogram below shows only the signal of the synthesized peptide. In this example, the purity of the peptide was calculated as 90%. Typically, the minimum purity threshold is 80% but some applications require purities >95%. In the peptide purity report, also the spectra of target compound and impurities typically truncations of the expected peptide sequence [3] are displayed (not shown here). This information can help to optimize the synthesis procedure. The visual representation of a 96-well plate in Fig. 4 shows some typical peptide QC results. Here, the left half of the spot indicates whether the expected peptide was found (green) or not found (red) whereas the color of the right half reflects the purity of the synthesized peptide. PDF result reports are stored on the Compass OpenAccess Server for data archiving and retrieval. Moreover, the reports are traceably linked to the respective raw data path information in case further data validation is required. The peptide purity method Figure 3: The peptide purity method provides the relative percentage content of the expected peptide in the synthesis product. Moreover, it gives information about the accompanying impurities (not shown here). Peptide QC result Figure 4: Peptide QC result visualization for a 96 well-plate in Compass OpenAccess.

6 Bruker Daltonics is continually improving its products and reserves the right to change specifications without notice. Bruker Daltonics , LCMS-112, Conclusion The described two-stage Compass OpenAccess QC workflow allows fast and automated quality control of synthetic peptides with minimal user intervention, thereby freeing laboratory staff for more productive and more complex tasks. In the first step, the identity of the synthesized peptides is verified at a theoretical throughput of 400 to 500 samples per day. Using this workflow the molecular mass of the expected peptide is automatically calculated even when uncommon amino acids or modifications are present. This removes the need for a manual and time-consuming calculation. Corresponding amino acid building blocks and modification tables are easily editable by the user. The second part of the peptide QC procedure provides information about the purity of the sample and whether the final product is acceptable or requires additional sample processing. Using this method, a throughput of samples per day is possible. References [1] Swietlow, A.; Lax R.: Quality control in peptide manufacturing: specifications for GMP peptides. Chem. Today 2004, 22, [2] Merrifield, R. B: Solid Phase Peptide Synthesis. The Synthesis of a Tetrapeptide. J Am Chem Soc 1963, 85, [3] Eggen, I.; Gregg, B.; Rode, H.; Swietlow, A.; Verlander, M.; Szajek, A.: Contol Strategies for Synthetic Therapeutic Peptide APIs Part III: Manufacturing Process Consideration. BioPharm International 2014, 27, For research use only. Not for use in diagnostic procedures. Bruker Daltonik GmbH Bremen Germany Phone +49 (0) Fax +49 (0) Bruker Daltonics Inc. Billerica, MA USA Phone +1 (978) Fax +1 (978) ms.sales.bdal@bruker.com -