Supplemental Figure Legends
Fig.S1 MALDI of His-16E1^E4 and phosphorylated His-16E1^E4
A. His-16E1^E4 was cleaved by in-gel digestion with chymotrypsin. The peptides were analysed by MALDI-TOF MS. Protonated ions of m/z (mass/charge) 2002 and 2789 were detected corresponding to the 16E1^E4 peptides GSTWPTTPPRPIPKPSPW (residues 17-34) and APKKHRRLSS DQDQSQTPETPATPL (residues 35-59) respectively. In the top of the figure, a diagram shows full length of 16E1^E4.
B, C and D. His-E1^E4 was phosphorylated in vitro with CDK1, CDK2, ERK respectively and non-radioactive ATP. The phosphorylated His-16E1^E4 were digested with chymotrypsin to produce smaller ions/peptides and analysed by MALDI-TOF MS. Addition of a phosphate group (HPO3-) leads to a mass increase of 80 Da (Dalton). Phosphorylation by CDK1 or CDK2 in 16E1^E4 gave a protonated ion of m/z ~2082, corresponding to addition of one phosphate to the peptide GSTWPTTPPRPIPKPSPW (B and C). ERK phosphorylation resulted in the ion of m/z ~2868, corresponding to addition of one phosphate to the peptide APKKHRRLSSDQDQSQTPETPATPL (D).
Fig. S2 Nanospray of CDK1 phosphorylated His-16E1^E4
A. His-E1^E4 was phosphorylated in vitro with CDK1 and digested with chymotrypsin. The samples were analysed by nanospray MS. A peak corresponding to the +2 form of the phosphorylated ion, GSTWPTTPPRPIPKPSPW (residues 17-34), was detected at m/z 1041.6 (GSTWPTTPPRPIPKPSPW with a mass of 2081 Da but with a charge of 2+, hence a m/z of 1041.6).
B. The ion of m/z 1041.6 was selected for collision-induced fragmentation in the ion trap. The spectrum shows the b and y ions detected after fragmentation. Above the spectrum is a diagram of the peptide sequence fragmented with the corresponding b and y ions aligned. The b14 ion (the subscript is the number of amino acids in the ion) of m/z 1516.9 corresponding to the unphosphorylated fragment GSTWPTTPPRPIPK and the y4 ion of m/z 566.1 corresponding to the phosphorylated peptide PSPW. This revealed that serine 32 (indicated in a box) was phosphorylated by CDK1. Another indication of the presence of phosphate is an ion of m/z 993 corresponding to the +2 ion of m/z 1041.6 losing a 98 Da phosphoric acid and then being ionised as a +1 form.
C. To confirm that serine 32 is the phosphorylation site, the y4 ion of m/z 566.1 was further fragmented in the ion trap. This produced a y3 ion of m/z 566.1 corresponding to the phosphorylated fragment SPW (residues 32-34) and an ion at m/z 468.1 corresponding to loss of phosphoric acid (98 Dalton) from PSPW, showing that serine 32 was the likely phospho-acceptor (shown in a box).