High-Quality Peptide Synthesis Platform

Understanding Your Peptide QC Report

Your peptide QC report consists of MS and HPLC analyses. The guide below will help you understand each analytical report, and how to tell if a quality analysis of your peptide was performed.

• HPLC report

• MS report

HPLC report

Even after multiple rounds of purification, small amounts of impurities may still exist in the final peptide product. Therefore, GenScript uses reversed-phase high performance liquid chromatography (RP-HPLC) to analyze the purity of each custom peptide.

What is RP-HPLC?

RP-HPLC is a widely used analytical tool, in which the components of a complex mixture can be efficiently separated. The analyte mixture is usually dissolved in water, which is sometimes also mixed with an organic solvent or an acid to assist dissolution prior to RP-HPLC analysis. The analyte is carried by a mobile phase consisting of water and an organic modifier, which is pumped through a column packed with a stationary phase which usually is a number of small diameter particles consisting of carbon chains of a specified length on the surface. As the mixture is pumped through the column, the analytes (in this case your custom peptide and small impurities) adsorb to the hydrophobic surface of the stationary phase. As the percentage of organic modifier in the mobile phase is gradually increased, the analytes desorb into the mobile phase. Desorption of a specific analyte is based on its intrinsic properties, thus each analyte will remain in the column for a specific amount of time, called the retention time. Since peptide bonds maximally absorb UV light at the wavelength of 220 nm, a UV spectrometer is commonly used in RP-HPLC to detect a peptide as it elutes from the column. The detection signal is converted to a visual graph called a chromatogram, which is the plot of UV absorbance vs. elution time.

High resolution of eluted peptides is mainly based on the selection of columns and the organic modifier, especially its elution gradient. The height of a theoretical plate of each column used in the manufacture is regularly checked, and different gradients are established to separate varying peptides. Both processes are aimed at maintaining high resolution of every custom peptide. GenScript takes special care in developing RP-HPLC protocols to ensure the most effective separation and the accurate purity determination of your custom peptides.

Reading Your Custom Peptide HPLC report

1. HPLC mobile phase and its elution gradient: The peptide separation is highly sensitive to the selection of organic modifier and its elution gradient.
2. Absorption wavelength: The wavelength of 220 nm is chosen for UV detection since peptide bonds (amide bonds) maximally absorb UV at this wavelength.
3. Column type and dimensions: The selection of the column plays an important role in RP-HPLC analysis. A C18 column is usually used to separate peptides.
4. Dead time: Indicates the amount of time required for an unretained solute such as the organic solvent or the acid from the column. Thus, the organic solvent nor the acid contributes to the area of the target peptide peak.
5. Major component elution peak (the target peptide): Shows the retention time of the target peptide as well as the impurities. The integrated peak area is used for the purity calculation. The baseline should be smooth, indicating that the system is stable. The vertical lines separate the partially overlaid peaks and denote the peak area of the target peptide.
6. Peak table: All peaks shown in the chromatogram are listed according to their retention time. The Area % of the target peptide is used to calculate the peptide HPLC purity.

MS Report

We use electrospray ionization mass spectrometry (ESI-MS) analysis to confirm the molecular weight of the target peptide.

What is ESI-MS?

ESI-MS is an analytical tool used to study macromolecules, where the mass-to-charge ratios (m/z) of all ionized analytes are respectively determined. As one of the widely used soft ionization techniques, electrospray ionization produces a whole charged ion from the solution of analytes without fragmentation of the macromolecule, which provides a reliable and accurate way to measure the molecular weight of the macromolecule. Generally peptides can be ionized into multiple-charged states, either positively or negatively, depending on the number of basic and acidic residues in the sequence.

The ionized peptides are then transferred into a mass analyzer such as a quadruple or a time-of-flight (TOF), which can separate all charged ions according to their m/z. All these isolated ions are respectively detected by an ion detector such as an electron multiplier and finally are converted into a visual graph called a mass spectrum, which is the plot of ion abundance vs. m/z value of all detected ions. The mass spectrum displays all detected m/z signals and the relative intensity of the ions, which is relative to the highest m/z peak. The highest peak is set as 100%. For most peptides, the mass spectrum displays more than one m/z peak representing a series of charge states. Theoretically the m/z values of these peaks should all result in the same calculated molecular mass, however, calculations from these peaks actually vary with each other slightly due to limited resolution of the instrument. In our MS report, the calculated molecular weight from the most abundant m/z peak of the analyte is utilized to largely eliminate variations from the instrument. GenScript performs regular cleaning and calibration of mass spectrometers to ensure the mass accuracy and reproducibility of MS analyses.

Reading Your Custom Peptide MS report

1. Ionization mode: Either positive or negative ionization mode can be used for the peptide analysis.
   Note: In positive ionization mode, the m/z value of the target peptide can be used to calculate the measured molecular weight (MW) by using the formula MW=(m/z)*IzI-z
2. z: +2, the measured MW=865.05*2-2=1728.10
3. z: +3, the measured MW=577.15*3-3=1728.45
4. z: +4, the measured MW=433.05*4-4=1728.20
5. Instrument settings: The optimized parameters for ESI ion source.
6. Measured MW by mass spectrometer: The MW calculated from the most abundant m/z peak of the analyte.

Quotations and Ordering

• For quotations, please use our Secure Instant Online Quotation/Order system. However, you may also contact us by email, phone (1-732-885-9188), fax (1-732-210-0262), or via our Secure Messaging System.
• Order can be placed with either a PO (Purchase Order) or credit card. Our customer service representatives are available 24 hours, Monday through Friday to assist you.
• Order can be placed with either a PO (Purchase Order) or credit card. We accept POs and major credit cards (). A 7% New Jersey sales tax will be applied to orders shipped to New Jersey. Your credit card will be billed under "GenScript" Click here to download our credit reference form. For international orders, we must apply the full charge at the time the order is placed. In the unlikely event that any given order cannot be filled, our guarantee will take the form of a full refund.

FAQs

• What quality control data is provided by GenScript?

  GenScript provides CoA, MS and HPLC validation data for each peptide. If other QC services are required, corresponding testing reports will also be provided.

• I would like to know what quality control measure you employ i.e., how do I determine that the product you sent me is what I wanted?

  GenScript's products and services are certified by ISO 9001:2015, the current and only valid version of the QMS standard. All GenScript peptides undergo Total Quality Management testing, including mass spectrometry(MS) and analytical high performance liquid chromatography (HPLC) analysis throughout the peptide synthesis and purification processes. These analyses are normally sufficient for custom peptide applications. 

• How to read my peptide QC report? Especially the HPLC and MS report?

  Please refer to this webpage for info: https://www.genscript.com/accupep_quality.html

• What is the best way to dissolve peptides?

  The solubility of a given peptide varies depending on its amino acid sequence and modifications. If there is solubility test requirement in your order, please dissolve peptide according to the solubility test report. If there is is no solubility test report, please refer to the CoA report for recommended solvent to disolve the peptide.

  It is difficult to know the exact solubility without a practice test, we recommend adding the peptide solubility test. By the way, if there is an exact requirement on the solvent, please kindly let us know and we can test it for you. If there is no solubility test in the order, you can refer to the recommended solvent in the COA report.

• How great an effect would the spacer(s) have on solubility?

  The effect spacer on peptide solubility depend on the different spacer types. Generally, there are three types, one type can optimize the solubility of the peptides in ultrapure water, such as PEG. One type can increase lipophilicity of the peptides, such as an alkyl fatty chain. The other type is neutral amino acid Gly, it is hard to confirm its impact on solubility, which may depend on a specific peptide sequence.

• The peptides were precipitated after further dissolved in 1xPBS to 0.3 mM from the stock of 10 mM in DMSO. May I know what I should do with these peptides?

  First, please check if the peptide could be dissolved in both DMSO and PBS, the solubility test report is needed to check whether the peptide could be dissolved in DMSO and PBS.

  For the peptide that already dissolved but precipitated, it is recommended to centrifuge the insoluble peptide and re-dissolve it in these soluble solvents according to the solubility test report.

• What kind of solubility test service can GenScript provide?

  Please refer to this webpage for info: https://www.genscript.com/peptide_solubility_testing.html

• What kind of solvent can be chosen in qualitative and quantitative solubility test services?

  Solvents that can be chosen in qualitative solubility test service (Cat No: SC1634, no more than 3 solvents) are as follows:
  Ultrapure water
  0.2M acetic acid
  1×DPBS (pH 7.1±0.1)
  0.1 M PBS (pH=7.4±0.1)
  DMSO (AR)
  8M Urea

  Solvents that can be chosen in quantitative solubility test service (Cat No: SC1634-1) are as follows:
  Ultrapure water
  0.2M acetic acid
  1×DPBS
  0.1 M PBS (pH=7.4±0.1)
  DMSO (AR)
  8M Urea
  Tris-HCl
  0.9% NaCl/Saline
  Other (can choose specific concentration of specific solvent)

• What is the meaning of “freely soluble”, “soluble”, “insoluble” in qualitative solubility test service? If GenScript could provide an exact concentration?

  Sorry, for qualitative solubility test service, GenScript cannot provide the exact concentration of the peptides in solvents. If needed, please order quantitative solubility test service. The results of qualitative solubility test service are according to the dissolution of approximately 0.5mg peptide in 0.5mL solvent.

  "Freely soluble": the peptide can be dissolved quickly and completely when the solvent is added to the peptide, and the dissolved peptide solution is clear and transparent.

  "Soluble": the peptide can be dissolved completely after sonication (no more than 30s), and the dissolved peptide solution is clear and transparent.

  "Insoluble": Precipitates occur when the solvent is added to the peptide even after sonication.

• How to proceed with the peptide if it shows “insoluble” in all chosen solvents in qualitative solubility test?

  GenScript will provide a soluble solvent for the peptide that is insoluble in chosen solvents in delivery COA report for your reference.

• How to proceed with the peptide that shows “freely soluble” in the report but cannot be dissolved?

  The result of “freely soluble” is based on the dissolution of 0.5mg peptide in 0.5mL solvent. GenScript recommends to add the solvents to achieve the concentration of 1mg/mL. If the peptide still cannot be dissolved, an ultrasonic instrument is recommended to be used.

• How can I obtain my peptide QC reports?

  For your reference, all your QC data can be downloaded from your account. Simply

  1. Log into your account

  2. Click "my orders/quotations" on the left panel

  3. Click the order ID you need, scroll down and you can find the “XX Data File(s)”, click it.

  4. Click the “ZipFile”, you can download all the reports you need.

  If there are any issues occur when downloading the reports, please feel free to contact GenScript’s project manager.

• What is the QC criterion of crude peptide? Would HPLC report be provided for crude peptides?

  HPLC reports are available for standard service peptides, and for the crude peptide library, there would be two options, one is “with only MS, no HPLC”, the other one is “COA, RP-HPLC, and MS for each peptide”. Then GenScript will respond according to your option.

  For “with only MS, no HPLC”, the crude peptide should meet the following condition before sending out:

  I. the peak with the highest relative intensity response in the MS report is the target peptide.

  For “COA, RP-HPLC, and MS for each peptide”, the crude peptide should meet the following conditions before sending out:

  I. the peak with the highest relative intensity response in the MS report is the target peptide;

  II. there is an obvious peak in the HPLC spectrum.

• Do you provide the Material Safety Data Sheet for us?

  Yes, we can provide material safety data sheet upon your request.

• How should I store my peptides?

  Generally, lyophilized peptides can be stored in a dry and tightly closed vial at -20 degrees, and be protected from intense sunlight. GenScript does not recommend storing excessive peptides in solution because the shelf life of peptides in solution is very limited, much shorter than peptides as lyophilized powder. This is especially true for peptide sequences that contain cysteine, methionine, tryptophan, asparagine and glutamine. It is necessary to aliquot the amount of peptide you need for the day and re-lyophilize the remaining peptide solution. Another drawback is that peptides stored in solution are susceptible to bacterial degradation. If storage of peptide in solution is unavoidable, use sterile buffers at pH 5-6 and store aliquots at -20 degrees; and pass the peptide solution through a 0.2 µM filter to rid the bacteria.