Fig. 3: Quantifying amino acids in neat patient plasma with bio orthogonal labelling.

a Our strategy involves performing five bioorthogonal labelling reactions. Each label is targeted to protein-incorporated amino acid residues of a specific amino acid type and reacts only with that amino acid type. Labels are fluorogenic, with fluorescence turning on exclusively after reaction with the targeted amino acid type of interest. Labelling is quantitative, so fluorescence intensity scales quantitatively with the concentration of the targeted amino acid type. b We identified fluorescence imaging regions where we did not observe autofluorescence from neat patient blood plasma. Fluorescence excitation spectra for labelling of protein-incorporated cysteine residues within the blood plasma of a patient with breast cancer, for the dye before mixing with the patient plasma, and for the patient plasma alone. c Because labelling is quantitative and no autofluorescence is observed from patient plasma, we use a calibration curve derived from solutions of known amino acid concentrations - Bovine Serum Albumin (BSA), Beta-Lactoglobulin (BetaLac), and Lysozyme (LYZ) of known protein sequence at known protein concentration — to determine the relationship between fluorescence intensity and amino acid concentration for each amino acid type. We used the quantitative linear fit to the protein calibration curve to transform the fluorescence intensity measured for cysteine labelling in the patient plasma samples into the corresponding concentration of cysteine amino acid residues. Data shown for triplicates of N = 20 breast cancer patients plasma samples. d The relationship between the experimentally measured Amino Acid Concentration Signature (AACS) with theoretically calculated AACS from the known concentrations of individual plasma proteins. The AACS of Tyrosine (Tyr), Cysteine (Cys_T) and Lysine (Lys) are shown in the graph. Data shown for triplicates of N = 20 healthy patient plasma samples. e To investigate whether changes in the host immune response to tumour development drove AACS alternations, we measured the AACS of mice which had been injected with an immunologically Hot Tumour or a Cold Tumour. Note: Throughout, Cys_R refers to free cysteine residues that are not disulfide bonded, while Cys_T refers to total cysteine residues, including those involved in disulfide bonding.