Laboratory Testing Blog

3 Tips to Overcome Challenges in Analyzing Peptides and Proteins by LC-MS/MS

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The recent increase in development of protein and peptide therapeutics demands highly informative methodologies.  More than 600 biological drugs are currently approved by FDA.  The interest in analyzing peptides and proteins arises from the need, during preclinical and clinical studies, to quantify levels of the metabolites in protein and peptide drugs.

Historically, large molecule quantitation was performed using ligand-binding assays (LBA’s).  LBA’s are associated with issues such as:

• Cross reactivity

• Limited linear dynamic range

• Long assay development times

• Poor accuracy and precision

This has led to the demand for a faster, more flexible, and more reproducible platform such as Liquid Chromatography-Mass Spectrometry (LC-MS).  Although LC-MS is the technique of choice for Bioanalysis, it is not without its challenges when it is being used for proteins and peptides quantitation.  In particular, handling issues such as non-specific binding, aggregation, poor solubility, and stability need to be addressed. In addition, chromatographic and mass spectrometric behavior of proteins and peptides is different from that of small molecules.

Finally, since these compounds are often modified or synthetic versions of biomolecules, separation, both LC chromatography and extraction/isolation must be fully optimized to ensure the highest degree of selectivity possible.  To support this challenging work, fast and flexible platforms for peptide quantification are needed that can deliver highly sensitive, reproducible and accurate results.  For last several years bioanalytical methods have been developed that provide accurate, selective and sensitive quantification of peptide and protein therapeutics.

For the purposes of bioanalytical method development, peptides can be distinguished from proteins based on size.  Peptides are classified with molecular weights of ~ 6,00Da or smaller.  Proteins comprise everything above this cutoff.

Immunoassay methods have an advantage because they are highly sensitive for large molecule detection, where MS/MS may not achieve adequate sensitivity.  LBA’s also require minimal sample preparation but suffer from poor linear quantification ranges and require antibodies.  From the point of view of a bioanalytical method, the most serious drawback of LBA’s is the interference observed from homologous peptides and high abundance proteins present in the samples and also poor selectivity between structural and chemically similar peptides or proteins.

LC-MS/MS detection mainly has significant advantages for quantification of peptides and proteins whenever selective antibodies are not available.  Other advantages including improved precision and accuracy.  Most importantly the resolving power of Ultra Performance Liquid Chromatography (UPLC) and specific MS/MS detection allows structurally or chemically similar peptides and proteins to be distinguished from each other.

Below are 3 tips that can help you overcome challenges in analyzing peptides and proteins by LC-MS/MS:

1. Sample Preparation

Protein precipitation provides the quickest and simplest technique for peptide analysis.  However, protein precipitation does not always provide a very selective clean up that removes only large protein interferences and it cannot be further optimized.  Failure to remove enough endogenous material during sample clean up can result in a matrix effect.  Where additional clean up and selectivity is required, solid phase extraction (SPE) can be adapted to overcome issues of poor selectivity, insolubility and protein binding.  The key to a selective methodology is through the use of SPE µelution plates.  The orthogonality of particles and modifications allow a simple yet flexible method of analyte extraction.  The µelution plates mean that precious analytes can easily be concentrated and labile peptides undergo the minimum of sample manipulation for high recovery and sensitivity.

2. Chromatographic Separation of the Analyte

ACQUITY UPLC Peptide Separation Technology (PST) columns provide the pore size required for larger molecules for accurate and precise retention characteristics – sharp, narrow UPLC peaks for accurate and precise quantitation.

3. Detecting the analyte with MS/MS

Using the UPLC-TQD tandem quadrupole mass spectrometer, a mass range of m/z 2000 on the first and second quadrupole allows the analyst to select the best charge state of the peptide to monitor for a high level of flexibility and simplicity of method development.  IntelliStart streamlines the process by selecting the best precursor and product ions, even when the product ion has a higher m/z than the precursor ion.

Combining the sensitivity of the TQD system with the selectivity and efficiency of SPE and UPLC, developing effective methods for peptide bioanalysis is significantly simplified.

Methodologies that overcome the challenges analyzing peptides and proteins in complex biological matrixes such as plasma and serum can be developed.  The above mentioned issues appear differently from one peptide to another and must therefore be addressed by the particular peptides assay method development.  However, there are some general guideline that can assist you with method development and achieving a robust, sensitive and specific method.  These provide a good starting point and have been successfully applied to developed and validated bioanalytical assays for peptides and proteins.