Figure 4. MS³ fragmentation data unravels the existence of two different phosphorylation sites.

(A-D) Representative spectra of FBP1 monomer purified from yeast grown in a glucose-containing medium. In general, individual proteoforms identified in the MS² spectrum (A) were selected within the quadrupole mass filter and subjected to MS³ fragmentation (B). The insets show fragment ion matches of two b ions, representing identified phosphopeptides. For each fragment, the position, charge and mass accuracy are indicated. Phosphorylated fragments are labeled by _p. In all generated MS³ spectra, fully resolved multiply charged fragment ions were detected using a mass resolving power of 140,000. (C-D) Selective MS² isolation of mono-phosphorylated or unmodified FBP1 proteoforms. For each proteoform selected, the respective MS³ spectrum is shown on the right panel of (C) or (D). Expansion of the 1,700-1,900 m/z region of MS³ data generated upon specific proteoform selection and fragmentation, showing the b16 ion, which matches the ²PTLVNGPRRDSTEGFD¹⁷ sequence. The data indicates that the b16 phosphopeptide (labeled in red) is exclusively found in the MS³ spectrum of the mono-phosphorylated FBP1 and not in that of the unmodified proteoform. (E) Analysis of MS² and MS³ spectra revealed that the FBP1 protein is missing the initial Met, and that it is phosphorylated at two mutually exclusive sites: at either position ¹²Ser/¹³Thr or within the stretch of ²⁴⁸Asn-³¹⁰Asp. In the latter, due to low fragmentation efficiency in this specific region, the exact phosphorylation position could not be mapped. b ions are indicated by brackets pointing towards the N-terminus and y ions are indicated by brackets pointing towards the C-terminus. Non-phosphorylated fragments are indicated in black, phosphorylated fragments are indicated in red. (F) A model of the FBP1 tetramer, demonstrating surface-exposed fragmentation hotspots. One subunit is colored in cyan and the other three are in different shades of gray. Fragmentation hotspot sequences are ³⁰HQKQFK³⁵, ⁷⁹DQQKKLDVLGD⁸⁹, ¹¹⁰QEDLIVFPT¹¹⁸ and ²²⁹WNETI²³³ (labeled by numbers 1- 4 respectively). The model highlights the structural organization of the complex, which is assembled from a dimer of dimers, in accordance to our results (Fig. S4).