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. Author manuscript; available in PMC: 2022 May 1.
Published in final edited form as: Proteomics. 2021 Mar 30;21(9):e2000140. doi: 10.1002/pmic.202000140

FIGURE 2.

FIGURE 2

Streamlined TMT workflow for SPS-MS3-based phosphoproteomics analysis. (A) Samples are lysed in 8 M urea-containing buffer with protease and phosphatase inhibitors. Proteins are isolated via precipitation, and proteolytically digested with Lys-C and trypsin. (B) The samples are labeled with TMT or TMTpro reagents in the digestion buffer (200 mM EPPS, pH8.5) plus 30% acetonitrile. These TMT labels act as a chemical barcode to track the origin of each protein from each sample. (C) Phosphopeptides are enriched by one or several methods. The flow-through from the enrichment (which is isobaric-tag labeled) can be used to profile the whole proteome of the sample under investigation. (D) Samples may be analyzed using SPS/RTS-MS3. The MS2 stage can include either lower Energy HCD (CE = 30–32) or multistage activation (MSA)-CID for peptide fragmentation to be analyzed in the ion trap. The MS3 stage consists of HCD-based fragmentation for Orbitrap-based mass analysis