Instrument Logic Increases Identifications during Mutliplexed Translatome Measurements.

Area of Science:

• Proteomics
• Biochemistry
• Mass Spectrometry

Background:

• Pulsed Stable Isotope Labeling in Cell culture (SILAC) is crucial for measuring protein dynamics like translation and degradation.
• Quantifying acute protein changes with SILAC is challenging due to low labeled peptide stoichiometry.

Purpose of the Study:

• To overcome limitations in pulsed SILAC quantification of acute changes.
• To introduce and validate a method using instrument logic and targeted mass differences (TMD).

Main Methods:

• Utilized instrument logic for targeted mass differences (TMD) to select specific labeled peptide ions.
• Compared standard data-dependent acquisition with TMD-enhanced acquisition for low-stoichiometry peptide mixtures.
• Evaluated both MS2 and MS3 methods for identification and quantification precision.

Main Results:

• Achieved 2-3 fold increases in peptide identification using TMD compared to standard acquisition.
• Maintained quantification precision across MS2 and MS3 methods with TMD.
• Demonstrated increased experimental throughput by reducing required instrument time.

Conclusions:

• Instrument logic with TMD significantly enhances pulsed SILAC for quantifying acute protein changes.
• This approach improves identification rates without compromising quantification accuracy.
• The method is broadly applicable to pulsed SILAC, with or without tandem mass tagging (TMT), for increased efficiency.