Fig. 1.

Identification of KLC1ser460 as a phosphorylation site by LC–MS/MS. (A) A schematic representation of the KLC1 structure showing the heptad repeats (green box) and the TPR domain (comprising six tandem repeats numbered 1–6). Serine 460 (S460) is located in a linker region between TPR domains 5 and 6. (B,C) Tandem MS/MS spectra of the KLC1 phosphopeptide ACKVDSPTVTTTLK fragment ion series, after collision-induced dissociation. (B shows the spectrum with transfected FLAG–KLC1 and C shows that with endogenous KLC1 isolated from rat cortical neurons). The m/z of the fragment ions is plotted against the intensity, and the detected ions of the b- and y-ion collision series are indicated. The pattern of fragment ions produced localises the phosphorylation site in both samples to serine 460. (B) The mass difference between y₈ and y₉ and the release of phosphoric acid from y₉ (y₉-H₃PO₄) is consistent with a phosphorylation on serine 460. [M+2H]²⁺ represents the peptide ion with m/z=800.9, used as the parent ion for fragmentation to produce the MS/MS spectrum; [M+2H-H₃PO₄]²⁺ indicates the neutral loss of phosphoric acid. (C) Neutral loss from b₆ ([b -H₃PO₄]²⁺) but not from y₁ to y₈ localises the phosphorylation site to serine 460. Consecutively matched y-ions without neutral loss indicate no phosphorylation of other potential sites (threonine 462, threonine 464, threonine 465 and threonine 466). The parent ion is absent following the fragmentation. [M+3H-H₂O]³⁺ at m/z=528.12 and [M+3H-H₃PO₄]³⁺ at m/z=501.39 represent the loss of water and the neutral loss of phosphoric acid from the parent ion, respectively.