Webinar Q&A follow up: ‘High-throughput mass spectrometric analysis of covalent protein-inhibitor adducts for the discovery of irreversible inhibitors: a complete workflow’

Automation MS (LC–MS/MS, HRMS, IMS) Sample preparation

Thank you everyone who attended the live webinar: High-throughput mass spectrometric analysis of covalent protein-inhibitor adducts for the discovery of irreversible inhibitors: a complete workflow. Below are responses to the questions posed during the live event that we did not have time to answer. We hope this is a useful resource and thank our webinar attendees and our speakers, Iain Campuzano (Amgen) and Vaughn Miller (Agilent Technologies), for their time.

Q&A follow-up

1. Do you add anything to reduce sodium adducts?

For the small proteins we have analyzed, which are in the 20-30 kDa Mw range, we do not see any significant sodiation. We are operating the ESI source Fragmentor Voltage at 200-225V for these proteins, which in combination with the 6 s load/wash cycle appears sufficient to remove salt adducts. For the larger proteins ~60 kDa, the levels of adducting was increased, but operating the Fragmentor at higher voltages, 275V in combination with extended load/wash times of 9-12 s appeared optimal for adduct reduction and minimization.

2. What factors determine relative % bound? How do you calculate this and is it reproducible?

Yes, POC, %-Bound and relative %-Bound are reproducible. We always make duplicate injections of the unmodified protein (same protein as used in the study) immediately prior to injecting the covalently modified protein samples.

The POC is calculated as follows: POC = (Unmodified Protein Peak Intensity/ Control Protein Peak Intensity) x100

%-Bound is calculated as follows: % Bound = (Expected Protein Adduct Peak Intensity/ Control Protein Peak Intensity) x100

Relative %-Bound is calculated as follows: Relative % Bound = % Bound/(POC + % Bound) x100

3. The human serum albumin (HSA) you show in one of the final slides, can that be used as a control protein?

Yes, the recombinant HSA can be used as an off-target control protein for certain reactive compounds of interest.

4. How many injections can you run on a single RapidFire cartridge?

The data shown in the presentation was over 1000 electrophilic compounds assayed by the C4-SPE RapidFire MS approach at 2 and 20 hr time points. So including wash steps and standard protein injections, that is over 2000 injections on a single C4-SPE cartridge.

5. How do you deal with the interference signal of the compound, which could be very dominant based on the concentration?

Currently we do not have a way of dealing with this issue in an automated fashion. As I indicated in the presentation, this interference can occur and can suppress the protein signal. These cases often result in an outlying data point (or points) in the correlation plots. So in these cases we would have to interrogate the data manually by C4-SPE integration and manual spectral deconvolution by MaxEnt 1. As described in our manuscript, our protein concentration is 2 uM and compound is either 10 or 100 uM depending on the particular issue we are addressing. With these conditions there have only ever been a few cases where the compound signal completely suppresses the protein signal. At much higher compound concentrations, this would be an increasing problem. As I also indicated on one of the questions, coupling this RapidFire-MS technology to an ion mobility enabled MS-system would help reduce interference by separating, in the gas-phase of the MS-system, the singly charged interference ions and the multiply charged protein ions of interest.