We have a Q-exactive Orbitrap mass spectrometer. I have noticed that the number of protein groups observed from 100 ng of HeLa whole cell lysate (purchased from Thermo) has gone down from 1800 to 1100. What could be a reason for decrease in the number of protein groups the instrument is able to identify?
Hi,
Can you explain what you mean by number of protein groups? Is that based on GO terms, for example?
There can be numerous reasons for why you find different data between different runs: did you analyze the exact same sample in both cases, or are these independent preparations of the same lysate? And how much time was in between the runs? Do you know how many peptides were detected in both runs, did you check what the TIC in both runs looks like, to get an idea of overall data quality comparability between runs? Have other people been running samples in the meantime?
Depending on the calibration of the MS, the overall cleanliness of the system, etc., the data obtained from a mass spec experiment can greatly differ.. A bit more information would be helpful, I guess.
Nameera Baig , you are using the pre-digested Thermo scientific Pierce HeLa Protein Digest Standard, consistent LC-MS-method and consistent data processing procedure? Are evaluation and calibration of your Orbitrap with Thermo-Cal-Mix done regularly and everything is fine? Most of the time my protein identifications decreased, I also observed a decreased normalized level and/or increased injection time while injecting cal-mix. In almost all of those cases our Orbitrap (Q Exactive HF) had to be cleaned by a Thermo technician. However, you could first check your "spray" in the source (visible droplet formation? -> bad -> exchange needle, or the column, if you are using EASY-equipment) and clean the ion transfer capillary + S-lenses, if you feel up to it. This would be my first-line-procedure.
Eef Dirksen, if Proteome Discoverer is used for data processing (which I assume), then a "protein group" is (roughly speaking) defined as a group of proteins, which all match to the observed peptides and which cannot be distinguished further based on the obtained mass spectra. Each group is represented by a "master protein", which is considered the most probable candidate of the group. Therefore, I think a "lower number of identified protein groups" is equivalent to a "lower number of identified proteins".
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