Different methods of protein analysis - characterisation of proteins and peptides

As proteins and peptides are complex molecules there are different needs for characterisation of proteins and peptides. There are different experimental such as detecting proteins, quantify proteins, for isolating and purifying proteins, and for characterizing the structure and function of proteins. However, some experimental methods (e.g., mass spectrometry) require computational analysis of the raw data.

Following we describe some of the approaches to analyse proteins or protein solutions.

Protein/Peptide Quantification Methods
Bradford

The Bradford protein assay is a spectroscopic analytical procedure used to measure the concentration of protein in a solution. The Bradford protein assay was developed by Marion M. Bradford, is based on an absorbance shift of the dye Coomassie Brilliant Blue G-250 in which under acidic conditions the red form of the dye is converted into its bluer form to bind to the protein being assayed. This very sensitive colorimetric assay has an absorptions maximum at 595nm and detection limit is 0.01mg protein/mL. Unlike other protein assays, the Bradford protein assay is less susceptible to interference by various chemicals that may be present in protein samples. An exception of note is elevated concentrations of detergent (e.g. SDS).

BCA
BCA (Bicinchoninic acid assay) is a spectroscopic analytical procedure used to measure the concentration of protein in a solution. The BCA protein assay was developed by Paul K. Smith, is based on the reduction on Cu2+ ions from the cupric sulfate to Cu+. The amount of Cu2+ reduced is proportional to the amount of protein present in the solution. Next, two molecules of bicinchoninic acid chelate with each Cu+ ion, forming a purple-colored product that strongly absorbs light at a wavelength of 562 nm. The detection limit is 0.005mg protein/mL. The test is very stable to most buffer and detergents, but small amount of reducing or chelating substances interfere with the test.

Amino acid analysis (P2145 >)
Employing quantitative amino acid analysis techniques enables a real quantification that is hydrolysing proteins or peptides with subsequent qunatification of each single amino acid by a chemical modification of each amino acid followed by LC separation and quantification by UV or fluorescence detection.

Intact Mass Determination
In order to determine the intact mass of proteins and peptides ESI-MS is employed using ion trap and high resolution FTICR mass spectrometry followed by charge state deconvolution.

Usual applications are
- confirmation of protein integrity (e. g. correct sequence length)
- evaluation of product heterogenity for biosimiliars
- detection of PTMs like phosphorylation or chemical modification
- indirect sequence validation.

Optionally proteins can be reduced and/or deglycosylated prior to analysis. As a standard, samples are ultrafiltrated/rebuffered into an MS compatible system and analysed by HPLC-ESI-MS to remove residual buffer components. Only pure, salt-free/detergent-free proteins can be analysed directly. Please consult us when your protein requires detergent to be stable in aqueous solution.

Please note that standard services are designed for the analysis of proteins with the following items in mind:

- purified protein is required, i. e. buffer must be defined. Industrial grade protein preparations often contain high amounts of impurities.
- protein should not require detergent to be kept in aqueous solution.
- high or especially complex glycosylation will necessitate treatment with PNGase F.

Please consult Genaxxon (info@genaxxon.com) in case your samples do not meet all requirements to discuss alternative sample preparation or analysis.

Protein/Peptide sequence information Methods
De novo sequencing
The determination of a protein's sequence can be necessary especially when no DNA data is available. This is especially true for
- proteins from non-common species where no genome data exists
- monoclonal antibodies when no hybridoma cells are avalable
- commercial proteins with undisclosed sequence

The aim of de novo sequencing is either to obtain peptide sequence tags for the generation of DNA primers or the elucidation of the full protein sequence, e. g. for the generation of biosimilars. To tackle such tasks, Genaxxon initially applies:
- different proteolytic digests of the protein
- high resolution mass spectrometry to generate high confidence data
- extensive bioinformatic analysis

If necessary further analyses can include:
- micropreparative HPLC of proteolytic peptides
- Edman sequencing of internal fragments
- experimental validation of results via synthetic peptides

As the scope and complexity of each sequencing project will vary by prior knowledge of the protein, its size and primary structure, please contact our specialists for consultation.

N-terminal sequencing (Edman degradation)
To elucidate the N-terminal amino acids of a protein, the classic step-wise chemical cleavage by automated phenylisothiocyanate chemistry as developed by Pehr Edman is a straightforward method. If you need to know the identity of a protein, have a look at protein identification by mass spectrometry which is more sensitive and applicable to a wider range of samples.
Genaxxon provides
N-terminal sequencing of proteins and peptides provided as lyophilized samples, in solution or blotted on PVDF membrane as fee-for-service. Proteins and peptides can be sequenced five to up to 40 steps depending on sample amount, sample purity, amino acid sequence and customer’s requirements.
Customer receive
A result report of the N-terminal sequence analysis with the detected amino acid sequence and all HPLC chromatograms as a PDF file by email.
Shipping details
- Please complete submission form for Edman sequencing with information about the samples (e. g. sample name, sample type, MW, protein amount, staining method) and the requested number of N-terminal sequencing steps.
- The samples should be in an Eppendorf reaction tube sealed with Parafilm. Protein samples on PVDF membrane can either be shipped as small and dry membrane slides in reaction tubes or as a dried PVDF membrane in plastic foil with enclosed description of the protein bands which should be analyzed. We cut the marked protein band for analysis.
- Liquid samples should be sent in frozen state.
- Lyophilized sample and samples blotted on PVDF membrane can typically shipped at room temperature.
- Send your samples in a padded envelope or in a box and enclose the sample submission form.
- Notify us of your order via email or phone, especially if you are shipping international.

Sample requirements

Sample amount

Minimum 25pmol. Please inquire if you have only smaller sample amounts available. We have a dedicated sequencer for small sample amounts (surcharge applies).

Sample type

1. lyophilized protein/peptide samples.

2. liquid protein/peptide sample in 10-100µl volatile solvent like Milli-Q water. isopropanol, acetonitrile. The sample have to be shipped in frozen state!

3. Protein samples blotted on PVDF membrane (PVDF membrane size: max. 3x6 mm, smaller and more concentrated sample are preferred. The protein band can be stained by Ponceau S, Amido black, Sulforhodamine or Coomassie Brilliant Blue.

Sample purity

The sample should be as pure as possible (at least 75-80% purity) and contain only the protein or peptide. Free amino acid, primary amines, SDS, salt, buffer or other contaminants should be as low as possible since the Edman chemistry can be negatively affected.

Cysteine modification

Cysteine without special modification can not be detected by N-terminal sequencing. Therefore the sample has to be modified for detection of cysteine. Below you can find a modification protocol.

N-terminal blockage

Proteins and peptides which are N-terminally blocked do not have a free N-terminal amino group. Therefore these proteins can not be sequenced. More than 50% of all eukaryont proteins are blocked. On request we can perform deblocking procedures but we need a significant higher amount of protein and the deblocking does not work always, because mostly the type of blockage is unknown.

Glycosylation and other modifications

Glycosylation and other modifications             Edman sequencing steps without the detection of an PTH amino acid, reduced peak intensity or altered retention times can be caused by glycosylation, phosphorylation or other modifications. Modified amino acids often cannot be sequenced. We recommend mass spectrometry instead

Our recommendations for protein blotting and staining
Chemical sequencing using Edman's phenylisothiocyanate chemistry targets the primary amino group on the protein's N-terminus. Accordingly buffers containing primary amines like Tris and glycine should be avoided for blotting as they significantly decrease sensitivity.
Semi-dry blotting
- PVDF membrane: Immobilon P membrane or comparable.
- Blot buffer: 50mM sodium borate, pH 9.0 / 20% methanol (HPLC quality) (0.1% SDS can be added to blotting buffer if protein above 40kDa should be sequenced).
- Blotting conditions: 1mA/cm2 PVDF membrane for 2-3 hours at +2°C to +8°C.
- after staining please wash membrane with water (Ponceau S) or 50% methanol/water (Coomassie).
Ponceau S (red) staining
- 0.25g Ponceau S
- 0.5mL acetic acid
- ad 50mL in Milli-Q water
Procedure:
1. Wash the PVDF blot membrane 2x 3 minutes with plenty Milli-Q water.
2. Stain the PVDF membrane with Ponceau S staining solution for 1-3 minutes.
3. Destain the PVDF blot membrane under visual control with Milli-Q water until protein bands are well visible.
4. Dry the PVDF membrane.
Coomassie Brilliant Blue R-250 (CBB R250) staining
Staining solution (0.1% CBB R250, 10% acetic acid, 40% methanol in Milli-Q water)

- 0.1g CBB R250
- 40mL methanol
- 10mL acetic acid
- ad 10mL in Milli-Q water
Destaining solution (10% acetic acid, 40% methanol in Milli-Q water):
- 40mL methanol
- 10mL acetic acid
- ad 10mL in Milli-Q water
Procedure:
1. Stain the PVDF blot membrane for 5 minutes in CBB R250 staining solution.
2. Destain the PVDF blot membrane for 3 x 5 minutes with destaining solution under visual control until protein bands are well visible.
3. Dry the PVDF membrane.
Sulforhodamine staining
Staining solution (0.005% sulforhodamine, 0.2% acetic acid, 30% methanol in Milli-Q water)
- 150mL methanol
- 1mL acetic acid
- 25mg sulforhodamine
- ad 500mL in Milli-Q water
Procedure:
1. Wash the PVDF blot membrane 2x 10 minutes with plenty Milli-Q water.
2. Dry the PVDF membrane at room temperature!
3. Stain the PVDF membrane in Sulforhodamine staining solution for 1-2 minutes.
4. Wash the PVDF membrane with Milli-Q water shortly.
5. Dry the PVDF membrane.
Alkylation of Cysteine residues
No signal will be obtained for Cysteine residues when they are not derivatised prior to analysis. Following amino acids will be detected normally, however. Two reagents are recommended for the alkylation of Cysteine thiol groups:
- iodoacetamide, a common reagent which is easy and safe to handle.
- 4-vinylpyridine, which gives superior signals in Edman sequencing.

Further reading: See the publication by Sechi et al "Modification of cysteine residues by alkylation. A tool in peptide mapping and protein identification >" for more information.

PTM Analysis (post- translational modifications)
Post-translational modifications are crucial in modulation of biological functions and thus are interesting targets for protein analysis. Other modifications can be introduced during recombinant protein production or subsequent purification and handling steps like advanced glycation end products (AGE) and others. They need to be monitored to ensure consistency of production batches.
As PTMs and other modifications cover a wide range of physicochemical properties, the strategies for their analyses can cover different techniques like electrophoresis, mass spectrometry and Edman sequencing. Please consult us to discuss your project's specific requirements.

Disulfide Bridge Analysis
Disulfide bridges can be determined by ESI-MS as the mass difference between 2 unbridged and 2 bridged Cys is about -2.0157 Da. Thus the existance of disulfides within a polypeptide of up to 20 kDa can be validated by determination of the intact protein mass by high-resolution ESI-MS. To validate single disulfides and correct pairing Genaxxon offers directed proteolysis under non-reducing conditions in order to generate suitable bridged peptides for mass spectrometric detection and fragmentation.

The price per analysis is dependent to:
- the number of disulfide bridges
- the need of proteolysis (single, multiple)
- verification of predicted disulfides vs analysis of all combinations

Terminal variants
Recombinant proteins are often existing as different protein species.
- N-terminal variants
- Lysine clipping (C-terminal variants)
- Internal truncation products
- N-terminal glutamine cyclization (-> Pyro-Glu)

According to the different variants of terminal variants different services to characterize terminal variants such as peptide mapping after directed proteolysis, N- & C-terminal sequencing and intact mass determination can be employed to analyse samples.
Further reading: Dick, L. W., Qiu, D., Mahon, D., Adamo, M. and Cheng, K.-C. (2008), C-terminal lysine variants in fully human monoclonal antibodies: Investigation of test methods and possible causes. Biotechnol. Bioeng., 100: 1132–1143. doi: 10.1002/bit.21855.

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