Compositions and methods for protein detection

A method of simultaneously detecting or quantitating multiple target p-hydroxyphenylpyruvate dioxygenase (HPPD) proteins in a complex biological sample from a crop plant comprising multiple target HPPD proteins and multiple non-target proteins, the method comprising:

• a) obtaining a biological sample from a crop plant;
• b) extracting proteins from the biological sample, resulting in an extract comprising a mixture of proteins;
• c) reducing an amount of insoluble proteins in the extract of step b), resulting in an extract of concentrated soluble proteins;
• d) digesting the soluble proteins in the extract of step c), resulting in an extract comprising peptide fragments, wherein the peptide fragments include an unlabeled surrogate peptide specific for each target HPPD protein of the multiple target HPPD proteins;
• e) concentrating the peptide fragments in the extract of step d),
• f) adding one or more synthetic labeled surrogate peptides comprising a label and an amino acid sequence of SEQ ID NO:
• 1 or 2 to the extract of step e) to create a peptide fragment mixture, wherein each of the one or more synthetic labeled surrogate peptides in the peptide fragment mixture is an analog of the unlabeled surrogate peptide specific for a target HPPD protein and has the same amino acid sequence as the unlabeled surrogate peptide specific for the target HPPD protein, and wherein a different synthetic labeled surrogate peptide is added for each of the multiple target HPPD proteins;
• g) concentrating the unlabeled surrogate peptides and the synthetic labeled surrogate peptides by reducing an amount of non-surrogate peptides in the peptide fragment mixture;
• h) resolving the peptide fragment mixture from step g) via liquid chromatography;
• i) analyzing the peptide fragment mixture resulting from step h) via mass spectrometry, wherein detection of a transition ion fragment of an unlabeled surrogate peptide and a transition ion fragment of the analogous synthetic labeled surrogate peptide is indicative of presence of the target HPPD protein that the unlabeled surrogate peptide is specific for;
• and optionally,
• j) calculating an amount of the target HPPD protein in the biological sample by comparing mass spectrometry signals generated from the transition ion fragment of the unlabeled surrogate peptide with mass spectrometry signals generated by the transition ion fragment of the analogous synthetic labeled surrogate peptide of the target HPPD protein.