## 3 Results  
### Identification of Novel Phosphorylation Sites in Human SMN  
Full-length human SMN comprises 294 amino acid residues. Here, we aimed to analyze phosphorylation of the C-terminus. Tryptic digestion of SMN resulted in long peptides, which could not be analyzed by mass spectrometry (MS). Therefore, we employed a strategy that would increase MS accessibility to the C-terminal region of human SMN (hSMN), whereby we introduced two trypsin cleavage sites into a hSMN-EGFP construct through site-directed mutagenesis (denoted as HH227, 273 RR; Figure 1A). The linker between SMN and EGFP introduced an additional arginine residue as a potential additional tryptic cleavage site. We then expressed this construct in differentiated motoneuron-like NSC34 cells and immuno-purified the mutated hSMN-EGFP protein with a GFPantibody bound to magnetic beads. After washing, we eluted the purified hSMN-EGFP fusion protein in reducing Laemmli buffer and analyzed its expression by SDS-PAGE and Coomassie staining followed by in-gel tryptic digestion (Figure 1B,C). To identify the protein composition of the gel band, 10% of the sample was analyzed directly by liquid chromatography MS/MS (LC-MS/MS) measurement. We detected hSMN-EGFP as the top protein with 27 unique peptides (\(p<0.01\)) and a protein coverage of 43% (Figure 1D). Interestingly, the C-terminal peptide \({}^{289}\)CSHSLNCR\({}^{296}\) of hSMN was also identified (of which C295 and R296 are part of the linker sequence; Figure 1D). This peptide contains two putative phosphorylation sites within hSMN (S290 and S292) as predicted by NetPhos 3.1 analysis [41]. We were able to detect both sites (\({}^{289}\)CpS\({}^{250}\)HSLNCR\({}^{296}\) and \({}^{289}\)CSHpS\({}^{292}\)LNCR\({}^{296}\)) after phospho-peptide enrichment with TiO\({}_{2}\) beads (Figure 1E). Together, we confirmed for the first time the putative phosphorylation residues hSMN S290 and S292 as novel SMN phosphorylation sites.  
Figure 1: Phospho-analysis by mass spectrometry identifies human survival of motoneuron (hSMN)  
S290 and S292 as phosphorylation sites. (**A**) Sequence alignment of hSMN wildtype (WT) and mutant hSMN HH227, 273 RR for trypsin cleavage at the C-terminus with the mutated site for additional trypsin cleavage sites shown in red. (**B**) The experimental workflow depicts the detection of phosphorylation sites in SMN. (**C**) SDS-PAGE and Coomassie staining of immunoprecipitated proteins. The box indicates the excised gel slice for MS analysis of SMN-EGFP. (**D**) LC-MSMS analysis after tryptic digestion identified hSMN HH227, 273 RR-EFGP (10% sample). Framed sequence: the detected non-phosphorylated peptide \({}^{289}\)CSHLSLNCR\({}^{296}\) comprising the C-terminus of hSMN HH227, 273 RR (289-294) and two amino acid residues of the EGFP tag (295-296). (**E**) ESI-MSMS identified of the co-eluting phospho-peptides\({}^{289}\)\({}_{\text{PR}}\)CP\({}^{290}\)HSLN\({}_{\text{PR}}\)CR\({}^{296}\) and \({}^{289}\)\({}_{\text{PR}}\)CSHpS\({}^{292}\)LN\({}_{\text{PR}}\)CR\({}^{296}\) after enrichment with TiO\({}_{2}\) (isobaric precursor ions _m/z_ 571.218; charge 2\({}^{+}\); PRC, propionamide of cysteine). Significant fragment ions for each peptide are indicated.