## 2 Results
### Determination of Phosphorylated Peptides in REF and SRPP  
Furthermore, 64 REF/SRPP protein spots in different 2-DE gels were further examined, using MS/MS analysis to determine their phosphorylated amino acids. They were positively identified as 23 protein isoforms in the REF/SRPP family. These protein isoforms belong to four unique SRPP/REF family proteins (named REF138, REF175, REF258, and SRPP204). Finally, 344 phosphorylated peptides were identified from different protein isoforms in the REF/SRPP family under different treatments, and they contain 138 unique phosphorylated peptides, which were identified from REF138, REF175, REF258, and SRPP204. Among them, 53, 8, 35, and 42 unique phosphorylated peptides were determined from REF138, REF175, REF258 and SRPP204, respectively. These peptides contain 129 phosphorylated amino acids and they were obtained from the 64 protein spots. These phosphorylated amino acids include 75 serine, 31 threonine, 11 lysine, 8 aspartic acid and 4 tyrosine (Figure 3 and Figure S3, Table S2).  
Six protein spots (S-50-53, 74 and 79) were determined as REF138, containing 53 unique phosphorylated peptides and 27 phosphorylation amino acids. The six protein isoforms were  
Figure 3: Phosphopeptides and phosphorylated amino acids in different REF and SRPP members. The changed pattern of different protein spots after the D24, D48, E24, and E48 treatments on the DIGE gels, phosphorylated peptides, phosphorylated amino acid sites, and the predicted 3-D structures for REF/SRPP family members REF138 (**A**), REF175 (**B**), SRPP204 (**C**), and REF258 (Figure S2) are highlighted. The capital letters and circles with different color represent the phosphorylated amino acids in different protein spots. The detail information for these peptides and phosphorylated amino acid sites is listed in Figure S3 and Table S2.  
detected from the six spots under four (D24, D48, E24 and E48) treatments, by a total of 24 MS/MS experiments (Figure S3, Table S2). Six protein isoforms were significantly up-regulated after E24 treatment, and five of them kept increasing with the elongated ethylene treatment (Table S2). With the elongated treatment, more phosphorylated amino acids were observed and a total of seven unique phosphorylated peptides were identified in the E48 sample. Six phosphorylated amino acids (Asp 4, Asp 6, Ser 43, Ser 81, Lys 128, and Lys 132) were examined in the E48 sample. Except for spot S52, the other spots had more phosphorylated amino acids upon ethylene stimulation, especially for spots 53 and 79 in the E48 treatment (Figure 3A). Ser 81 was phosphorylated under both the E24 and E48 treatments, but it was not a phosphorylated amino acid in D48 (Figure S3). Only 8 phosphorylated peptides with 6 phosphorylated amino acid sites were observed in the two REF175 spots (Figure 3B). However, the phosphorylated amino acids decreased after ethylene stimulation, indicating that the phosphorylation of REF175 might be not as important as in the other REF members.  
Five spots (S-20, 21, 23, 24, and 27) were identified as REF258, containing 35 phosphorylated peptides with 16 phosphorylated amino acids that were detected from the five spots under four (D24, D48, E24, and E48) treatments, by a total of 20 MS/MS experiments (Figure S3, Table S2). They were sharply up-regulated after being treated with ethylene for 24 h, but three spots (S-23, 24 and 27) decreased in the E48 treatment (Table S2, Figure S3). Seven spots were identified as SRPP, among them, SRPP204 was the most abundant (Table S1). Three abundant SRPP204 spots, namely, S31, S49, and S70, which were induced by ethylene stimulation, were further selected to detect their phosphorylated peptides. The three SRPP204 isoforms contained 42 phosphorylated peptides with 24 phosphorylated amino acids. These results revealed that large amounts of peptides were phosphorylated in SRPP204, and 24 amino acids were phosphorylated in the detected three SRPP204 isoforms (Figure 3C, Figure S3, Table S2).