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. Author manuscript; available in PMC: 2015 May 6.
Published in final edited form as: Cell Metab. 2014 Apr 24;19(5):836–848. doi: 10.1016/j.cmet.2014.03.023

Figure 2. The translation initiation codon of PTENα is identified by MALDI-TOF mass spectrometry and terminal analysis with de novo sequencing.

Figure 2

(A) A pET28a plasmid containing PTENα with a C-terminal His-tag used for in vitro purification and mass spectrometry sequencing.

(B) His-selected affinity purification of PTENα. Bacteria-expressed His-PTENα was purified using nickel affinity chromatography. Combined fractions containing the slow migrating PTEN band (fractions 6-10) confirmed by PTEN immunoblotting (not shown) were separated with SDS-PAGE. Mass spectrometry analysis of a purified ∼70 kDa band (in red box) revealed six pieces of peptide that match the 5′-UTR region of PTEN, including a peptide near the CTG513-leucine site.

(C) The MS/MS spectrum of the peptide (GGEAAAAAAAAAAAPGR) that matches the N-terminus sequence of PTENα.

(D) A pFastBac1 plasmid containing PTENα with a C-terminal His-tag used for in vitro purification and mass spectrometry sequencing. The PTEN ATG start codon was mutated to ATA to avoid PTEN co-purification with PTENα.

(E) Purification of SF9-expressed PTENα with a C-terminal His-tag (band in red box) forMALDI-TOF-TOF MS and TMPP-Ac-OSu derivatization for de novo sequencing.

(F) Tandem spectrum of m/z 989.3727 in TMPP-Ac derivatized PTENα.

(G) De novo analysis of m/z 989.35 showing the first amino acids of PTENα, Leucine-Glutamic acid-Arginine (LER).

(H) Generation of Pten C-terminal FLAG knock-in mice for verification of the Ptenα gene locus.

(I) Verification of expression of FLAG-tagged PTEN and PTENα in Pten-FLAG knock-in mice. Liver and lung tissues from Pten-FLAG knock-in mice or control wild-type mice were lysed for immunoblotting with anti-FLAG antibody.

(J) Protein lysates of Pten-FLAG knock-in liver tissues or control tissues were subjected to sequential immunoprecipitation with anti-FLAG M2 agarose and a PTENα-specific antibody. The bound proteins were separated with SDS-PAGE and gel slices at around 70-kDa were analyzed by mass spectrometry. Four peptides were identified in Pten-FLAG knock-in tissues that match the N-terminally extended region of PTENα, αN.

See also Figure S2.