Peptide Quality

1. What purity of peptide do I need for my research?
2. What Quality Control (QC) information do you provide?
3. How pure are your catalog peptides?
4. What is the difference between “research-grade” and “GMP-API”?
5. What is the purity level for crude peptide?
6. Do I have to expect batch to batch variability?
7. What are the impurities of my peptide?
8. What is gross peptide weight?
9. What does net peptide content mean?
10. What is the difference between peptide content and peptide purity?
11. How do you calculate theoretical net peptide content?
12. What is a counterion?
13. Can you explain the [M+Na] and [M+K] mass peaks observed in the MALDI-TOF or ESI mass spectra?
14. How do you confirm a peptide is cyclized?
15. Why do my crude peptides have a bad odor?

What purity of peptide do I need for my research?

AAPPTec provides custom peptides in the following purities:

1. Immunological Grade (suitable for forming polyclonal antibodies)
2. 80% or greater (tissue culture; ligand for affinity purification; non-quantitative antibody blocking experiments)
3. 90% or greater (in vivo studies; bioassays; markers for electrophoresis; monoclonal antibodies)
4. 95% or greater (ELISA; RIA; enzyme substrate)
5. 98% (NMR; chromatography standards)

What Quality Control (QC) information do you provide?

Quality control documentation provided with every peptide includes mass spectral and HPLC analyses determining composition and purity. Amino acid analysis is available upon request. We also provide storage and handling guidelines.

How pure are your catalog peptides?

The HPLC-purity of most AAPPTec peptides is 95% or above. Labile peptides, e.g. peptides containing free Cys or an N-terminal Gln, or sulfated peptides, and “difficult” peptides may have lower purity.

What is the difference between “research-grade” and “GMP-API”?

“Research-grade” peptides are intended for laboratory and research purposes only!

They may not be used as drugs. GMP-API’s are sterile products monitored for chemical and biological contaminants applicable to humans. All AAPPTec catalog peptides are research grade unless specifically noted otherwise.

What is the purity level for crude peptide?

Generally the purity of crude peptides is >60%. However, depending on the difficulty of the sequences, this estimation cannot be guaranteed for all cases.

Do I have to expect batch to batch variability?

The impurity profile and especially the peptide content can vary somewhat from batch to batch, even if the protocols of synthesis and purification have not been changed.

What are the impurities of my peptide?

The impurities are mostly incorrectly synthesized peptide fragments/deletions, incompletely de-protected peptides and residual water.

What is gross peptide weight?

The gross peptide weight is the weight determined after weighing the peptide. This is the amount indicated on the product label.

What does net peptide content mean?

The gross weight of dry peptide doesn’t consist of peptide only, but includes non-peptide components such as water, absorbed solvents, counter ions and salts. Net peptide content is the actual percent weight of peptide from the gross weight. This number may vary, from 50-90 percent, depending on the purity, sequence and method of synthesis and purification.

What is the difference between peptide content and peptide purity?

Peptide content is not an indication of peptide purity; these are two independent measurements. Purity is determined by HPLC and indicates the presence/absence of contaminating peptides with undesired sequences. Net peptide content only gives information on the percent of total peptide versus total non-peptide components independently of the presence of multiple peptides. Net peptide content is accurately found by performing amino acid analysis or UV spectrophotometry. This information is important when calculating concentrations of peptide during sensitive experiments.

How do you calculate theoretical net peptide content?

Theoretical net peptide content (calculated assuming that counterions are the only non-peptide components present in your peptide sample) can be estimated by dividing molecular weight of the peptide by a sum of this molecular weight and a number of trifluoroacetate counterions that are required to neutralize the peptide multiplied by the molecular weight of the TFA counterion (MW= 114). For example, a synthetic peptide of MW=1000 with a free N-terminal amino group and one Arg has theoretical net peptide content of 1000/(1000 + 2 x 114 ) = 1000/1228 =0.81 or 81%. In practice, counterions are not the only possible contaminants in the peptide sample. It can also contain water, absorbed solvents and traces of other substances. As a result, the actual net peptide content is usually determined by quantitative amino acid analysis.

What is a counterion?

Most peptides except those that do not have basic amino acids such as Arg, Lys, His or those with blocked N-termini exist in the form of their salts. Synthetic peptides that are purified by HPLC are usually obtained as TFA salts. Their basic amino acid residues and N-termini are protonated and have trifluoroacetate (CF3COO -) counterions.

Can you explain the [M+Na] and [M+K] mass peaks observed in the MALDI-TOF or ESI mass spectra?

Peptide molecules can be ionized by the Na+ or/and K+, it is common to see their adducts from the MALDI-TOF or ESI mass spectra. Those ions come from the water used for the peptide purification or the buffer used for the mass analysis. They can never be completely removed, even if distilled or deionized water is used. Instead of being ionized by a proton, some peptide molecules are ionized by Na+ or/and K+ ions. It is common and normal to observe the [M+Na] and [M+K] peaks in MALDI-TOF mass spec.

How do you confirm a peptide is cyclized?

There are two common types of cyclizations that can be performed:

1. N-terminal to C-terminal cyclization
2. Disulfide bridge cyclization

N-terminal to C-terminal cyclization is confirmed by a molecular weight shift of 18 mass units in the mass spectrum. A disulfide bridge cyclization is confirmed by MS and HPLC before and after the cyclization step. Although a mass shift of 2 mass units can be difficult to detect for certain peptides, an HPLC shift helps confirm the completion of the reaction.

Why do my crude peptides have a bad odor?

If your peptide(s) have a thiol odor, it is because thiol scavangers were used during peptide cleavage and deprotection to prevent undesired side reactions. Although the peptide product(s) are thoroughly washed with ether to remove the thiol compounds (procedure below), minute traces (parts per billion) of thiols remain causing an odor. If the peptide is further purified by HPLC, most of the odor will be removed.

Peptide Cleavage Procedure

1. Place the resin in a round bottom flask and add 20% piperidine in DMF until the resin is just covered. Let the mixture stand for 30 minutes to remove the N-terminal Fmoc group.
2. Transfer the resin to a sintered glass funnel with fine porosity and apply vacuum. Wash the resin 3 times with DMF. Slurry the resin in DCM three times to remove the DMF.
3. Slurry the resin in 85%TFA in DCM (v/v) containing water, thioanisole, ethandithiol, and methyl disulfide to scavange cations released from side chain protection groups. Swirl the mixture occasionally during the reaction time. The reaction time will depend on the amino acid composition of the peptide.
4. Filter the resin in a fine sintered glass funnel. Wash the resin 3 times with small portions of TFA.
5. Combine the filtrates and add 8-10 times the volume of cold ether. If necessary, keep the mixture at 4°C overnight to precipitate the peptide. Isolate the precipitated peptide by filtering with a fine sintered glass funnel or by centrifuging. Wash the crude peptide ten times with cold ether to remove scavengers. Odor from the scavengers may still remain unless additional HPLC purification is performed.