Compact mass spectrometry in modern SPPSDaniel Eikel of Advion and James Cain of Protein Technologies showcase two examples of a new analytical technique in hard-to-synthesise peptides*

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Solid-phase peptide synthesis (SPPS) allows for the rapid and parallel synthesis of peptides of various amino

acid lengths and can range from picomole library generation to the bulk delivery of hundreds of millimoles of target compound. Modern SPPS systems provide flexibility between different synthesis chemistries, including the standard Fmoc-tBu and Boc-Bzl approaches, and can be automated up to the level of single-button operation.

However, challenging amino acid combinations, longer peptide targets and large-scale peptide production require reaction optimisation and careful parameter selection for improved synthetic yields at every cycle of the synthesis.

Cycle monitoring allows immediate control of the progression of SPPS reactions and can help to minimise these problems, leading to cost-efficient synthesis of the desired peptide. Also, like other chemical reactions, SPPS requires adequate quality control of the final peptide or protein product, including amino acid (AA) sequence confirmation.1,2,3

Mass spectrometry (MS) has been shown to be a very powerful analysis tool for peptides

and proteins and is routinely used in proteomics and in antibody and small molecule synthesis applications. MS can provide not only the molecular mass information of the SPPS cycle product but also AA sequence and structural information through specific and selective molecule fragmentation along the AA backbone.4,5,6

A recent development in MS is the design of smaller systems, with reduced vacuum pumping requirement but full analytical capability, called compact mass spectrometry (CMS). CMS systems with fragmentation analysis capabilities are therefore very cost-effective for analysis and ideally suited for reaction and quality control in SPPS, including detailed product characterisation.7

Here, we evaluate the utility of a system comprising an automated peptide synthesiser, the Prelude X from Protein Technologies, and Advion’s LC-CMS analysis system Expression-L, based on two peptides selected for their known synthetic challenge: the 65-74 position peptide of the acyl carrier protein (65-74ACP) and the Jung Redemann peptide from the AKR/Gross MuLV virus (JR-10mer).8

The two model target peptides are both challenging to synthesise. They were chosen to monitor synthesis progression along the SPPS cycles and characterise the final product quality obtained from SPPS.

Methods & material65-74ACP has the amino acid sequence

VQAAIDYING (Val-Gln-Ala-Ala-Ile-Asp-Tyr-Ile-Asn-Gly) with a theoretical mono-isotopic mass of Miso(65-74ACP) = 1062.53 (free carboxylic acid and amine on the C and N terminus respectively).

The ACP peptide was synthesised on the Prelude X at 20 μmol scale using Fmoc-Gly Wang resin (loading 0.4 mmol/g). Deprotection was performed with 20% piperidine in DMF for twice 30 seconds at room temperature (RT). Couplings were performed at a final concentration of 50 mM AA (10 eq.), 50 mM HCTU (10 eq.) and 100 mM NMM (20 eq.) for two times one minute. The cleavage cocktail used was TFA/H2O/TIS and the cleavage reaction was performed for two hours at RT.

JR-10mer is derived from the c–terminal portion of the AKR/gross murine leukemia

Figure 1 – LC-CMS chromatogram of final 65-74ACP product synthesis cycle

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virus (MuLV) that triggers a specific cytotoxic T-lymphocyte (CTL) immune response. Its AA sequence is WFTTLISTIM (Trp-Phe-Thr- Thr-Leu-Ile-Ser-Thr-Ile-Met) with Miso(JR-10mer) = 1210.63 (C terminus amide and free amine N terminus).

The JR-10mer peptides were synthesised on the Prelude X at 50 μmol scale using rink amide resin (loading 0.32 mmol/g). Deprotection was performed with 20% piperidine in DMF for one minute at RT. Couplings were performed at a final concentration of 250 mM AA (10 eq.), 250 mM HCTU (10 eq.) and 500 mM NMM (20 eq.) for two minutes at RT (first synthesis) and 90°C (second synthesis). The cleavage cocktail used was TFA/anisole/H2O/EDT and

the reaction was performed for two hours at RT. Samples were obtained either as lyophilised

peptide cleaved from the resin (as Fmoc-protected cycle product) or as lyophilised final peptide product released from all protection groups. Cycle products were dissolved in formic acid for further analysis; final SPPS products were dissolved in water with 0.1% formic acid at 0.4 mg/ml prior to CMS analysis.

Mass spectrometry analysis was performed using an Advion LC-CMS system consisting of a gradient pump, a Kinetex 2.6 micron XB-C18 100A 50 mm x 2.1 mm HPLC column from Phenomenex and an Expression-L CMS. A ten-minute gradient run from 5% B to 50% B in seven minutes was run at 800 µl/minute

with an MS scan range from 100 to 2,000 m/z at a rate of 7,500 m/z units/s. Solvent A was 0.1 vol % formic acid in water and Solvent B was 0.1 vol % in methanol.

ResultsThe final product of the 65-74ACP SPPS

was analysed by LC-CMS with 2.1 µg of the material on column (Figure 1). The total ion chromatogram (TIC) shows one dominant peak at 2.37 minutes elution time, with other minor components from 1.5-3.0 minutes in the ten-minute gradient run.

The extracted ion chromatogram (XIC) of the theoretical m/z 1063.5 shows the peak at 2.37 minutes to have the experimental mass-to-charge ratio of m/z 1063.55. This was a good first indication that this peak was indeed the intended 65-74ACP peptide.

However, only the additional in-source fragmentation data at the same elution time can confirm the analyte of interest. Here, the entire b-ion series, starting with b2, is marked in the mass spectrum and overlaps with the experimental data (Figure 2); the labels show the respective amino acid, some y-ion series are present as well but not labelled, theoretical values calculated using the Institute for Systems Biology’s Fragment Ion Calculator.9 With the additional in-source fragmentation data, the final peptide product can unequivocally be determined to be the intended 65-74ACP peptide product in both mass and AA sequence.

A similar analysis of the JR-10mer final SPPS product was performed and showed a significantly more complex LC-CMS chromatogram, suggesting the formation of more than one product and a significant amount of by-product. To better understand the SPPS and guide optimisation efforts, we also analysed each cycle of this SPPS by removing some resin material after each cycle and analysing the growing peptide chain while still in its Fmoc-protected state.

Cycles C4 to C9 (Figure 3) showed the growing of the peptide chain. All through cycle 6, the MS data shows the expected SPPS outcome. However, when adding the first threonine to the chain (cycle 7, top right), there is a significant amount of unreacted peptide left. Adding a second threonine in cycle 8 again shows a significant portion of unreacted peptide, now from two synthesis steps.

Continuing to cycle 9, the addition of phenylalanine appears to be normal, but the phenylalanine is added to all products from steps 7 and 8 and therefore results in a mixture of at least three major peptides, with one or both intended threonine amino acids missing. We could therefore point to the threonine addition in cycles 7 and 8 as the yield-limiting step of this SPPS that causes both the loss of product yield as well as the unintended by-products observed before.

Optimisation of this synthesis step resulted in a much improved second SPPS with a final

Figure 2 – MS data shown for the identical elution time with activated in source fragmentation (Note: b-ion series of the 65-74ACP VQAAIDYING marked with red lines, respective amino acids added for clarity)

Figure 3 – CMS data from the respective Fmoc protected SPPS cycle peptide of JR-10mer from cycle 4 (top left) through 9 (bottom right).

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product LC-CMS showing the predominant analyte to be the intended peptide (Figure 4, XIC on bottom trace). In-source fragmentation of the signal in this retention period shows almost the entire b-ion series of the JR-10mer peptide (Figure 5) and confirms the signal at RT 4.74 minutes as the product signal.

ConclusionModern SPPS shows a high degree of

automation and its relative simplicity has promoted its use in many laboratories, both for small-scale peptide library development and large-scale target production.

However, some peptide targets remain challenging to synthesise and even a single sub-optimal synthesis cycle can cause costly re-runs in large-scale or long-chain protein production. In addition to a full peptide product characterisation, including sequence, structural and purity information of the SPPS product, it seems logical to include a reaction control step after each synthesis cycle to guide yield optimisation efforts and ensure quality control.

MS is uniquely suited to support this analysis need because it can provide both the analyte mass-to-charge ratio as well as structural and AA sequence information, owing to specific peptide fragmentation patterns induced through, for example, in-source fragmentation. In addition, recent instrumentation development resulted in a new class of CMSs that are small enough in size to be placed bench-side to the SPPS, are cost-effective because of reduced vacuum pumping requirements and at the same time show the full analytical capabilities of modern MS.

Based on the two example peptides above, we have shown the monitoring and final product confirmation of the ten-cycle SPPS of both peptides and can conclude that the cost-effective LC-CMS analysis tool is ideally suited to support SPPS analytical needs.

Dr Daniel EikelProduct Manager, Ion SourcesAdvion, Inc.Tel: +1 607 266 9162E-mail: deikel@advion.comWebsite: www.advion.com

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1. L. Raibaut, O. El Mahdi & O. Melnyk, Top. Curr. Chem. 2015, 363, 103-542. P.R. Hansen & A. Oddo, Methods Mol. Biol. 2015, 1348, 33-503. V. Mäde, S. Els-Heindl & A.G. Beck-Sickinger, Methods Mol. Biol. 2014, 10, 1197-2124. P. Roepstorff & J. Fohlmann, Biomed Mass Spectrom. 1984, 11(11), 6015. K. Biemann in J.A. McCloskey (ed.), Methods in Enzymology 193, Academic, San Diego, 1990, 886–8876. K. Biemann, Biomed. Environ. Mass Spectrom. 1988, 16(1-12), 99-1117. X. Bu, Y.Yang, X. Gong & C.J. Welch, J. Pharm. Biomed. Anal. 2014, 94, 139-448. T. Redemann & G. Jung in R. Ramage & R. Epton (eds.) Proceedings of the 24th European Peptide Symposium, Mayflower Scientific, Kingswinford, UK, 1998, 7499. http://db.systemsbiology.net:8080/proteomicsToolkit/FragIonServlet.html

References:

Figure 4 – LC-CMS chromatogram of second SPPS synthesis of Jung Redemann 10-mer after optimisation of SPPS cycle 7 & 8

* – Also contributing to this article were Elisabeth Restituyo-Rosario of Protein Technologies & Simon J. Prosser of Advion

Figure 5 – In-source fragmentation CMS data at the elution time of JR-10mer (Note: b-ion series of the JR-10mer WFTTLISTI marked with red lines, respective amino acids added for clarity)

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