Internal Standards for Protein Quantification by LC-MS/MS

Introduction

The increased development of biopharmaceuticals makes a growing demand for the sensitive and reliable quantification determination of proteins in complex biological matrices such as plasma and serum. The method for quantifying proteins in biological matrices is based on ligand binding assays (LBAs) before. Currently, liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) has been demonstrated that can be used for the quantification of proteins. One of the main strengths of the technique lies in the possibility to use internal standards that correct for different sources of analytical variability. Furthermore, analytical methods using this technique can be set up and validated in a relative short period of time of typically a few weeks. Noteworthily, when using this technique, the use of an internal standard is usually necessary. Here, Alfa Chemistry describes the different internal standards used in the field of quantitative bioanalysis of protein according to the previous reports.

Internal Standards in Protein Quantification

A stable-isotope labeled form or a structural analogue of either the intact protein or the signature peptide can be used as internal standard for quantitative determination of protein. In addition, a modified form of the stable-isotope labeled peptide containing one or more cleavable groups is a possibility that also can be used as internal standard. The details are as follows^[1]:

Fig. 1. The workflows of protein quantification
by using different internal standard.

Stable-isotope labeled proteins as internal standards: To produce stable-isotope labeled protein internal standards, heavy isotope labeled amino acids are incorporated into the process of cell culturing, and the organism will incorporate the heavy isotope label in its proteins, thus creating the stable-isotope labeled protein of organism that is subsequently purified and used as an internal standard. An alternative approach is production of the stable-isotope labeled protein internal standards in a cell-free system containing a lysate of Escherichia coli or wheat germ. A stable-isotope labeled version of the protein can be added to the sample at the very beginning of the analytical procedure as internal standard to correct for all sources of variation throughout the entire analytical procedure.
Structural analogue proteins as internal standards: A structural analogue of the protein of interest can also function as internal standard. To select the internal standard candidates, candidates should exhibit a close resemblance to the protein of interest to sufficiently correct analytical variability during all sample handling step. Factors to consider include molecular mass, isoelectric point, number of chargeable amino acids and the release of a suitable peptide upon digestion. This approach has the advantage that one can choose from many commercially available proteins, often of high purity and for a reasonable price, eliminating the need to synthesize a specific protein especially for this purpose. However, such an internal standard will not correct completely variability for all of the sample preparation steps.
Stable-isotope labeled peptides as internal standards: When a stable-isotope labeled protein is unavailable, a stable-isotope labeled peptide can be used as internal standard which is added to the digestion mixture. For stable-isotope labeled peptide internal standards, any one or a number of amino acids in the sequence can be replaced using the corresponding stable-isotope labeled amino acid. This internal standard will not correct for steps in the procedure preceding and including the actual release of the signature peptide from the protein during digestion. It will however correct for variations caused by the extraction steps and the final LC–MS/MS analysis.
Stable-isotope labeled peptides containing a cleavable group as internal standards: The internal standard molecule consists of a stable-isotope labeled version of the signature peptide to which a cleavable sequence tag has been attached. This tag is cleaved during trypsin digestion releasing the stable-isotope labeled peptide. Thus, in addition to correcting for any subsequent sample handling and instrumental analysis steps, this approach offers partial correction for variations in trypsin digestion efficiency.
Structural analogue peptides as internal standards: An analogue peptide may also be used as internal standard. Of all internal standard approaches, this is the least attractive because it provides no correction for protein extraction and digestion and only limited correction for peptide extraction and instrumental analysis. For this reason, this approach is now rarely used.

Alfa Chemistry is a global leading supplier of analytical chemistry reagents. We provide a wide range of high quality internal standards and many other analytical chemistry products. If you cannot find the suitable products, Alfa Chemistry also offers you with custom synthesis service. If necessary, please don't hesitate to contact us.

Reference

Bronsema K. J., et al. Internal standards in the quantitative determination of protein biopharmaceuticals using liquid chromatography coupled to mass spectrometry[J]. Journal of Chromatography B, 2012, 893: 1-14.

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