Custom peptide synthesis Standard | modified | heavy peptides | libraries 2 Contents

Custom peptide synthesis 4

An overview 4

Standard peptide synthesis service 6

PEPotec Immuno Peptide Libraries 7

Targeted quantitation 8

HeavyPeptide and LightPeptide AQUA standards 9

PEPotec SRM Peptide Libraries 11

3 Custom peptide synthesis An overview

Optimizing the efficiency of peptide synthesis can be challenging. At Thermo Fisher Scientific, we have the experience, equipment, and knowledge to meet your needs for custom peptide synthesis. Our synthesis team based on your input. Our experienced peptide scientists has accumulated significant expertise through successfully will support you with peptide sequence, scale, and/or producing tens of thousands of custom peptides. We are purity selections for your assay to help you achieve the constantly adapting our product offerings to your needs best results for your application (Table 1).

Custom peptide synthesis capabilities

Table 1. Summary of peptide services and specifications. HeavyPeptide and PEPotec immuno PEPotec SRM LightPeptide AQUA Standard peptides peptide libraries peptide libraries Standards Primary Basic research or biomarker Epitope mapping, vaccine Quantitative mass Targeted proteomics application discovery development spectrometry Over 400 modifications High quality; accurate and Key features High throughput, low toxicity High throughput available precise concentration Peptide length* 2–110 amino acids 6–20 amino acids 6–25 amino acids Up to 15 amino acids Manufacturing 1 mg–1 g ≥1 mg ≥0.1 mg Multiple scales available scale** Crude (as synthesized) or Purity Crude (>50%) to >98% Crude (as synthesized) >95% to >97% >70% MALDI-TOF MS check or Quality control MALDI-TOF MS and HPLC MALDI-TOF MS analysis; HPLC for purified MALDI-TOF MS, HPLC, AAA grade 2–3 weeks AQUA Express, Production time 2–3 weeks 4 weeks 2–3 weeks 5–7 weeks standard AQUA * Please inquire about longer peptide lengths. ** Greater amounts available upon request.

Quality control Delivery format Quality control for our peptide services includes analyses Our peptides are provided in lyophilized and liquid formats using mass spectrometry (MS) for peptide identification, in glass vials as well as in 96-well Thermo Scientific™ analytical high-performance liquid chromatography (HPLC) Matrix™ tube plates, depending on the product for peptide purity, and amino acid analysis (AAA) for category. Please inquire for additional formats and other peptide concentration. Additional nonstandard analyses are custom requests. available on request. Analysis results are reported on the Certificate of Analysis for your records.

4 Professional support Peptide applications We have experienced and dedicated staff to assist you The use of synthetic peptides has significantly impacted through peptide selection, ordering, tracking, and delivery many areas of research. Our portfolio of custom peptide processes. We realize that the process can be lengthy, and services supports a variety of end applications, and we we strive to keep you informed every step of the way. strive to provide high-quality, fit-for-purpose peptides to enable success.

Peptide services

Custom peptides Peptide libraries Heavy peptides Unmodified PEPotec Immuno Peptide Libraries HeavyPeptide AQUA Modified PEPotec SRM Peptide Libraries LightPeptide AQUA

13 15 R( C6 N4)

Alexa Fluor dye label R

Applications

Protein interactions Quality control testing Assay development Quantitative proteomics ELISA, enzymatic, etc. SRM, PRM, SureQuant Assays

Epitope mapping Structural studies

5 Standard peptide synthesis service

High-quality peptides from validated Modifications process tailored to help meet your needs We offer a wide range of N-terminal, C-terminal, and other The Thermo Scientific™ Standard Peptide modifications with our standard peptide service (Table 3). Synthesis Service offers numerous options Please go to thermofisher.com/peptides for the most up- for peptide purity, modifications, and formats, to-date list and available positions in the peptide sequence. providing you the flexibility to meet your research needs. Custom conjugation services are also available, including conjugation to carrier proteins (KLH, BSA, OVA, or Thermo Our peptide synthesis utilizes the latest Fmoc Scientific™ Imject™ Blue Carrier™ Protein) and MAPs. solid‑phase technology and reversed-phase HPLC for Table 3. Peptide modifications offered with standard peptide purification. Peptide sequence is confirmed by MS, peptide synthesis. and peptide purity is determined using analytical HPLC. • Acetylation of N terminus (Ac-NH–) • Fluorescein (FITC/5-FAM) • Acetyl-lysine • Hydrocarbon spacers Highlights • Aldehyde • Hydroxy proline • High quality—all peptides are synthesized with • Invitrogen™ Alexa Fluor™ dyes • Hydroxy tryptophan high‑quality materials • 6-amino hexanoic acid (Ahx) • Isotopically labeled amino acids 13 15 • 6-amino caproic acid (Aca) (with C, N) • Modifications—most comprehensive list of available • Mercaptopropionic acid • Amidation of C terminus (–CONH2) modifications and labels • Amino benzoic acid • Methoxy-coumarin-acetic acid (MCA) • Beta-alanine • Validated—all peptides are analyzed by MS alone or in • Methionine sulfone • Biotin combination with analytical HPLC • Methionine sulfoxide [Met(O)] • Carbamido methylation (CAM) • Monomethyl lysine • Citrulline • Flexible—peptides available in a variety of formats and • Monomethyl arginine • Conjugation to proteins (BSA, KLH) purities; manual synthesis available for difficult peptides • Multiple antigen peptides (MAPs) • Conjugation to oligonucleotides • Norleucine (Nle) • Custom FRET peptides Table 2. Specifications for standard grade peptides. • Phosphorylation of serine, • Cyclization via termini or disulfide threonine, and tyrosine bridge • Polyethylene glycol (PEG) spacer Peptide length* 2–110 amino acids, L or D isoforms • D amino acids • Pyroglutamic acid (Pyr) Manufacturing 1 mg –1 g • Dabcyl scale** • Rhodamine • Dabsyl Purity Crude (>50%) to >98% pure • Special amino acids (D amino • Dansyl acids, other amino acids) Counterion TFA • Dihydroxy tyrosine • Tetramethylrhodamine (TAMRA) Formulation Lyophilized • Dimethyl lysine • Invitrogen™ Texas Red™ dye Delivery format Glass vial • Dinitrophenyl (DNP) • Ubiquitination Production time Standard delivery within 2–3 weeks • EDANS • Other dyes or modifications on Shipment Lyophilized, room temperature • Farnesyl request MALDI-TOF MS for crude peptides; MALDI-TOF MS and Quality control HPLC for all other purity grades Extensive list of modifications and labels, including Table 4. Peptide purity recommendations Modifications heavy peptides (isotope-labeled) by application. Custom conjugation available, e.g., protein-peptide Conjugation conjugates and multiple antigen peptides (MAPs) Alternate counterion, AAA, endotoxin testing, Crude: peptides for high-throughput screening Optional services solubility testing >70%: antigens for polyclonal antibody production * Please inquire about longer peptide lengths. >75%: ligands for affinity purification, enzyme substrate studies, epitope mapping ** Greater amounts available upon request. >80%: qualitative peptide blocking studies, protein electrophoresis applications >85%: immunological applications, peptide arrays, affinity purification >90%: qualitative bioassays, monoclonal antibody production >95%: quantitative bioassays (e.g., ELISA, enzymology, biological activity) >97%: MS proteomics applications >98%: structural studies, crystallography, NMR, in vivo applications thermofisher.com/standardpeptide Note: Listed purity levels indicate the minimum typical requirements for each application.

6 PEPotec Immuno Peptide Libraries

Ideal for epitope mapping and high-throughput Thermo Scientific™ PEPotec™ Immuno Peptide Libraries are screening for immunology applications fully synthetic custom libraries that support high‑throughput screening assays to map epitopes or identify immunogenic sites in proteins. These libraries are fully customizable and supplied with acetate as the counterion to avoid potential toxicity issues in cell-based applications.

Table 5. PEPotec Immuno Peptide Library service.

Peptide length* 6–20 amino acids; L isoforms only Quantity ≥1 mg Purity** Crude (as synthesized) C-terminal residue Any unmodified L isoform amino acid Counterion Acetate Formulation Lyophilized Peptides are provided in individual 2D barcoded tubes in Delivery format Highlights Matrix 96-tube plates • Low toxicity—acetate used as the counterion to reduce Production time 4 weeks toxicity when compared to trifluoroacetate (TFA) [1] Shipment Lyophilized, room temperature Minimum order 4 peptides; surcharge for orders of <48 peptides • Convenient—lyophilized peptides provided in individual Quality control*** MALDI-TOF MS 2D barcoded tubes in 96-tube plates Acetylated Lys, carbamidomethylated Cys, Modifications phosphorylation, etc. • Flexible—optional services, including phosphorylation Optional services Solubilization in DMSO, individual tube labeling, etc. and acetylation, are available for studying signaling and * Please inquire about longer peptide lengths. ** No purification given, as peptide purity is sequence-dependent and a function of different liquid regulatory proteins chromatographic conditions. *** For all peptides except phosphopeptides, the major peak in the MS analysis represents the target Applications of interest. Peptides must pass both the MS analysis and the final gross weight criteria (>1 mg) before shipment. If peptides do not pass these criteria, the customer will be informed and one resynthesis • Epitope mapping of B and T cell will be offered free of charge.

• Vaccine development

• High-throughput peptide screening

• Biomarker discovery

• Cell signaling (kinase/protease studies)

Reference 1. Pini A et al. (2012) Efficacy and toxicity of the antimicrobial peptide M33 produced with different counter-ions. Amino Acids 43:467–473.

thermofisher.com/pepotec-immuno

7 Targeted quantitation Quantitation of selected proteins

While discovery proteomics globally provides relative We offer products that enable assay development from quantitation for entire proteomes, targeted proteomics validation to quantitation, including Thermo Scientific™ SRM focuses on the quantitation of specific proteins with high Peptide Libraries and Thermo Scientific™ HeavyPeptide™ precision, sensitivity, and selectivity. and LightPeptide™ AQUA Standards for quantitation.

The goal of targeted quantitative analyses is to Targeted quantitative analyses require fast, robust, and investigate selected protein targets from discovery or cost-effective platforms, and both HRAM Orbitrap mass hypothesis‑driven studies, to understand biological spectrometers and triple quadrupole mass spectrometers pathways or to verify and select biomarkers from multiple are ideally suited for quantitative analysis. Triple quadrupole biological sources and conditions. mass spectrometers identify and quantitate peptides by serially monitoring specific mass windows for peptides of Synthetic peptides are an integral part of targeted assay interest, isolating and fragmenting, followed by quantitation. design and development. The traditional targeted assay This selective reaction monitoring (SRM) strategy for experimental design begins with selection of proteotypic targeted quantitation, along with chromatographic retention peptides through software-assisted selection or utilization time information, provides high sensitivity and specificity. of data from discovery proteomics experiments. These HRAM Orbitrap instrumentation quantifies proteins using selected peptides are then often inexpensively screened parallel reaction monitoring (PRM). A PRM assay is using a crude peptide library. After optimal peptide different from an SRM assay in that all fragmented ions sequence selection, highly pure heavy peptides of the from a selected precursor ion are monitored, rather than best candidates are synthesized and purified for target a selected fragmented ion from a selected precursor ion. quantitation. The heavy peptides serve as internal As a result, the ability to analyze all ions via PRM results in quantitative standards for absolute quantification of the higher specificity and faster development time, but with a corresponding natural peptides in a biological sample. trade-off in sensitivity.

8 HeavyPeptide and LightPeptide AQUA standards

Improve quantitative precision for absolute quantitation

AKT1 peptide 1

Standard Calibration Curve 102 Slope: 8.9630E-2 Intercept: 9.4231E-4 R-Squared = 0.9958 101 Weighting: 1 / (x * x)

10 0

10-1

10-2 Light:Heavy Peak Area Ratio The HeavyPeptide and LightPeptide AQUA custom 10-3 synthesis services provide isotopically labeled or unlabeled 10-2 10-1 10 0 101 102 103 AQUA (Absolute QUAntitation) peptides with extensive QC Analyte Concentration (fmol) for relative and absolute quantitation of proteins. IGF1R peptide 1 Standard Calibration Curve HeavyPeptide and LightPeptide standards are synthesized 102 Slope: 1.0508E-1 Intercept: 3.1858E-3 using the latest Fmoc solid-phase peptide synthesis R-Squared = 0.9962 101 Weighting: 1 / (x * x) technology, purified by HPLC and analyzed by mass spectrometry. Amino acid analysis (AAA) provides the most 10 0 exact peptide concentration to guarantee the highest level 10-1 of quantitative precision. The different grades of AQUA 10-2

peptides are defined by the replicates of AAA, with Basic Light:Heavy Peak Area Ratio (1X), QuantPro (2X), and Ultimate (3X). 10-3 10-2 10-1 10 0 101 102 103

Analyte Concentration (fmol)

Highlights Target Peptide no. LOD (fmol) LLOQ (fmol) ULOQ (fmol) Linearity (R2) 1 0.008 0.031 200 0.9958 • Precise—peptide concentration guaranteed from AAA AKT1 2 0.008 0.031 200 0.9872 1 0.008 0.031 200 0.9962 IGF1R • Sensitive—enables absolute quantification of 2 0.008 0.125 200 0.9664 low‑abundance proteins (fmol) Figure 1. HeavyPeptide AQUA analysis. Heavy peptides were • Specific—100% peptide sequence specificity selected from discovery MS data. Serial dilutions of light AQUA peptides and constant amounts of heavy AQUA peptides were analyzed in a ™ ™ ™ • Flexible—variety of purity, modification, and 6-protein digest matrix using a Thermo Scientific Dionex UltiMate 3000 RSLC Nano System (300 nL/min, C18 reversed-phase column) and formatting options Thermo Scientific™ Q Exactive™ HF Hybrid Quadrupole-Orbitrap Mass Spectrometer. Each serial dilution sample was analyzed by targeted Applications PRM-MS. Data were analyzed using Skyline software to generate 6-point calibration curves, and the results for figures of merits are summarized in • Biomarker discovery, • Confirmation of RNA the table. verification, and validation interference (RNAi)

• Functional quantitative • Pharmacokinetics proteomics • ADME toxicology studies • Quantitation of posttranslational • Anti-doping testing modifications

thermofisher.com/heavypeptide

9 Table 6. Specifications of HeavyPeptide AQUA-grade standards. AQUA Ultimate Service AQUA QuantPro Service AQUA Basic Service

Confirmation or validation, ideal for Discovery, confirmation or valida- Research stage Validation, ideal for absolute quantitation biomarker verification tion, relative quantitation 10 nmol/10 aliquots 10 nmol/10 aliquots Amount/number 40 nmol/40 aliquots 40 nmol/40 aliquots 15 to 30 nmol (0.05 to 0.1 mg)/1 aliquot of aliquots 96 nmol/96 aliquots 96 nmol/96 aliquots

Formulation 5 pmol/µL in 5% (v/v) acetonitrile/H2O 5 pmol/µL in 5% (v/v) acetonitrile/H2O Lyophilized Shipment In solution on wet ice In solution on wet ice Dry at room temperature Concentration ±5–10% ±10 –25% NA precision Peptide length* Up to 15 amino acids Purity >97% Isotopic >99% enrichment Standard (light) Yes peptides available Delivery format Glass vial

Production time 5–7 weeks standard AQUA

Quality control MALDI-TOF MS, HPLC, quantitative AAA

Single or double phosphorylation (pY, pT, or pS) Cysteine carbamidomethylation (CAM) Modifications Methionine sulfoxide [Met(O)] Other modifications available on request

Additional light amino acids to extend peptide length Optional services Additional heavy amino acids within the peptide sequence Multiple solvents, concentrations, and aliquot sizes available

* Please inquire about longer peptide lengths.

Table 7. Heavy amino acids offered with HeavyPeptide Custom Synthesis.* Amino acid Code Mass difference Isotope Isotopic enrichment

13 15 Alanine A +4 Da U- C₃, N >99%

13 15 Arginine R +10 Da U- C₆, N₄ >99%

13 15 Isoleucine I +7 Da U- C₆, N >99%

13 15 Leucine L +7 Da U- C₆, N >99%

13 15 Lysine K +8 Da U- C₆, N₂ >99%

13 15 Phenylalanine F +10 Da U- C₉, N >99%

13 15 Proline P +6 Da U- C₅, N >99%

13 15 Valine V +6 Da U- C₅, N >99% * Other amino acids on request.

10 PEPotec SRM Peptide Libraries

Fully synthetic, crude peptides customized for the development of medium- to high-throughput SRM and PRM assays

The study of proteomes, subproteomes, and protein pathways often requires quantitative MS analysis that depends on the development and validation of SRM, MRM, and PRM assays. The PEPotec SRM Peptide Libraries offer great convenience and fexibility for the development of quantitative MS with many customizable options.

Highlights Applications • Traceable—peptides are provided in individual 2D • Medium- to high-throughput development of SRM, barcoded tubes in 96-tube plates MRM, and PRM assays

• Customized—various grades with optional • MS workflows with relative and absolute services available quantitation strategies

• Convenient—delivered solubilized in 0.1% trifluoroacetic acid (TFA) in 50% (v/v) acetonitrile/water thermofisher.com/pepotec-srm

• Flexible—extensive list of available modifications

PEPotec SRM Peptide Libraries—four grades to fit your experimental needs. Grade 1 Grade 2 Grade 3 HPLC >70% pure Fast and easy Greater analysis Maximum assurance Guaranteed purity

Peptide length* 6–25 amino acids Quantity ≥0.1 mg C-terminal residue** Lysine (K) or arginine (R) Counterion TFA Formulation Suspended in 0.1% TFA in 50% (v/v) acetonitrile/water Delivery format Peptides are provided in individual 2D barcoded tubes in Matrix 96-tube plates Production time 2–3 weeks Shipment In solution on wet ice Single or double phosphorylation (pY, pT, or pS) Cysteine carbamidomethylation (CAM) Methionine oxidation [Met(O)] Modifications Internal heavy amino acids Alternative heavy amino acid at the C terminus Other modifications available on request Lyophilization Optional services Individually labeled tubes 24 peptides; surcharge for 4 peptides; surcharge for 4 peptides; surcharge for 4 peptides; surcharge for Minimum order orders of <48 peptides orders of <48 peptides orders of <48 peptides orders of <48 peptides You pay for entire set of You only pay for peptides You only pay for peptides You only pay for peptides Failed synthesis policy peptides ordered successfully synthesized successfully synthesized successfully synthesized Peptide resynthesis Not provided Not provided One resynthesis provided One resynthesis provided Peptide amount and MS Peptide amount, MS spectra, Included documentation Peptide amount Peptide amount spectra HPLC Purity Crude (as synthesized) Crude (as synthesized) Crude (as synthesized) >70% (guaranteed) MALDI-TOF MS check of MALDI-TOF MS check of MALDI-TOF MS analysis of MALDI-TOF MS and HPLC Quality control 5% of peptides 100% of peptides 100% of peptides analysis of 100% of peptides *Please inquire about longer peptide lengths and manufacturing scale **Other stable isotope labeled amino acids are available, please inquire.

11 To contact us by email or to send the quote form:

• Europe, Middle East, and Africa: [email protected]

• Rest of world (including North America): [email protected]

Find out more at thermofisher.com/peptides

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