Absolute Protein Quantification by Mass Spectrometry: Not as Simple as Advertised

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Accurate quantification of proteins is crucial in biological and clinical research.
• Bottom-up proteomics assays rely on calibration strategies using standards for reliable results.
• Stable isotopically labeled (SIL) materials are commonly employed as calibrators or standards.

Purpose of the Study:

• To evaluate the suitability of different SIL materials (peptides and proteins) for calibrating human thyroglobulin (Tg) quantification.
• To assess the commutability of SIL peptides and proteins as calibrators in bottom-up protein analysis.
• To investigate the accuracy and potential biases in protein quantification using various calibration methods.

Main Methods:

• Comparison of SIL tryptic peptides, cleavable SIL peptides, and full-length SIL proteins as internal calibrators and standards.
• Quantification of three forms of human thyroglobulin (native, commercial, recombinant) using bottom-up analysis.
• Standardization of all SIL materials and human proteins by amino acid analysis for traceability.

Main Results:

• Tryptic and cleavable SIL peptides showed a lack of commutability as internal calibrators across different bottom-up assay conditions.
• Recombinant protein calibrators can lead to inaccurate concentration assignments of native proteins due to variable digestion efficiency.
• Digestion efficiency variations were not resolved by altering denaturation/digestion stringency.

Conclusions:

• SIL peptides are not commutable and should not be used as internal calibrators in bottom-up protein assays.
• Recombinant SIL proteins are superior to SIL peptides as internal standards for quantifying human-derived proteins.
• The findings highlight potential inaccuracies in 'absolute' protein quantification using peptide or protein calibrators in bottom-up assays.