Liquid chromatography coupled mass spectrometry (LC-MS) operating in targeted mass spectrometry (MS) including multiple reaction monitoring (MRM) mode is an accurate analytical method. Integrating with nanoelectrospray-tandem MS (NanoES-MS/MS) and immunoprecipitation (IP), it becomes a promising method in detecting serum biomarkers which are at extremely low concentrations, such as mycobacterium tuberculosis (Mtb) secreted 10 kDa culture filtrate antigen (CFP-10). We employed an optimum denaturing condition for trypsin digestion and IP of targeted peptides. The condition shows several advantages over commonly used one, 1) it prevents further dilution of sample, thus enables as-needed sample volume, 2) It ensures liberation of trace amount of antigen peptide under a cost-effective enzyme to substrate mass ratio, 3) It avoids time-consuming and unnecessary reduction of disulfide bonds, as some pathogen antigens don't contain disulfide bond. More importantly, optimum MRM transitions of the targeted peptide and standardized data processing and interpretation algorithm provide confirmative information of it in serum samples. By changing the denaturing buffer, addition of a clean-up step, selection of optimum MRM transitions and better interpreting MS signal, this workflow showed an improved accuracy in clinical samples.

Sample pre-treatment by denaturing buffer 1. 100 µL of serum was processed in a 1.5 mL of centrifuge tube, after mixing with 900 µL of denaturing buffer and heated at 100℃ for 5 minutes using heat block. Depending on the total volume of serum sample that needs to be processed, the volume of denaturing buffer as well as the number of aliquots can be scaled up with the dilution factor unchanged. In our setting for Mtb antigen detection, the sample volume was 200 µL. Therefore, two aliquots were prepared for each sample. Caution! When moving the samples from heat block, it is suggested to wear thermal insulation gloves. 2. The denatured samples is cooling down to room temperature by sonicating them for 3-7 minutes in water bath. Once a number of samples are sonicated, the tubes needs to be placed in a suitable holder, i.e., Eppendorf Mixmate 1.5/2.0 mL. This will prevent leakage and unexpected contamination. 3. The pH of sample is adjusted to ~8.5 by adding 20 µL of 1M Tris buffer. Mix the samples thoroughly through vortex, and spin down all solvent that remains on the tube lid. Protein digestion 4. After mixing of 10 µg of sequence grade trypsin into each tube, the samples are placed on a Hulamixer® and incubated with rotation for 16 hours. The speed for rotation of mixer can be changed based on the number of samples that are placed on. To prevent bubbles which may influence the digestion efficiency, it is better to rotate in a mild speed without tilting. 5. The pH of sample is adjusted to ~7 by adding 10 µL of 10% trifluoroacetic acid (TFA) buffer. Mix the samples thoroughly through vortex, and spin down all solvent that remains on the tube lid. Caution! After adding TFA, there is white precipitate appeared in the bottom of tube. It is normal and needs to be clarified by vertex. Binding of peptide antibody to the protein G magnetic beads 6. The optimum amounts of magnetic beads and antibody vary among different peptide targets. We already listed potential peptide targets for Mtb infection detection (Table 1). For the CFP-10 peptide, they were determined to be 3 mg beads and 50 µg peptide antibody. When binding them together, 400 µL of binding buffer is added, and the beads and antibody are incubated for 1 hour with rotation on Hulamixer® at room temperature. To achieve the best binding efficiency, the amount of binding buffer and the time for binding can be adjusted according to the antibody used. Before binding, the magnetic beads need to be washed with 200 µL of PBS one time to remove any buffer remnant. After binding, the beads need to be washed with 200 µL of binding buffer twice to remove excess antibody. 7. The beads are re-suspended in 400 µL of binding buffer and stored at 4℃ until use. Typically, the beads can be stored for no more than two weeks in order to maintain the activity of antibody. IP of targeted peptide 8. Determination of the optimum amount of beads and antibody. For each tube containing 100 µL of serum, 0.15 mg magnetic beads and 2.5 µg peptide antibody are used. We obtained this optimum amount of beads/antibody by setting a serial of conditions with varied amounts and compared the MS signal for a constant amount of spiked CFP-10 protein. For another antigen, this optimum amount of beads/antibody needs to be determined prior to testing of clinical samples. 9. After adding the optimum amount of beads, the samples are incubated for 1 hour with rotation on Hulamixer® at room temperature. 10. The beads are combined from two aliquot and re-suspended in 200 µL of PBS buffer. 11. The beads are transferred into a new centrifuge tube and washed with 200 µL of PBS buffer twice, followed by 100 µL of LC grade water one time. 12. The captured peptides are eluted from the beads by incubating the beads with 100 µL of 1% formic acid buffer for 30 minutes at room temperature. The beads are placed on Hulamixer® with rotation to prevent them sinking to the bottom of tube. The eluent is separated from beads by magnet. Clean-up of IP eluent by StageTips 13. The StageTips packed with C8 disk are conditioned with 50 µL of 0.1% TFA in acetonitrile followed by 50 µL of 0.1% TFA in water. To remove liquid from them, the tips are placed in an adaptor filled centrifuge tube and centrifuged at 20℃, 2,000g for 3 minutes. Caution! Due to the changed height of tube with StageTips, the rotor lid cannot be attached during centrifuge. Place the tubes into the centrifuge in an ordered way so that the lids of tubes won’t contact with the inner wall of centrifuge. 14. The IP eluent is loaded into the StageTips from their top end using pipette. The StageTips are centrifuged under the same condition in step 12. 15. To elute the peptides, 50 µL of 0.1% TFA in acetonitrile is added into each tip and the tips are centrifuged under the same condition in step 12. 16. All solvent is evaporated under vacuum. To prevent sample loss, reconstitute the samples immediately using 1% formic acid. 17. After centrifuging the reconstituted samples at 20 ℃, 21,000g for 15 minutes, the supernatant is transfer into an MS sample vial. Caution! Prevent contacting the bottom of tube and picking up any invisible particle by leaving one µL of solution. MRM analysis of the targeted peptide 18. To set up the LC and MS methods for a targeted peptide, it is better to prepare a various concentrations of internal standard of that peptide solutions and get the gradient of organic solvent and collision energy optimized based on the MS signal of these solutions. The gradient of organic solvent and MS parameters used here is listed in Document for LC-MRM method. 19. After loading the sample onto LC column, the MS spectra are collected using the optimum MS method setting.

Healthy donor serum samples are purchased from Valley Biomedical (http://www.valleybiomedical.com/), the patient serum/plasma are collected from several sites according to their IRB approved protocols.