TABLE 2.

Isolation Efficiency of Phosphopeptide and Subsequent Dephosphorylation Verification Performed on Gyrolab MALDI IMAC1 Followed by Analysis Using Two Different MALDI Mass Spectrometers

Summary	CDs Analyzed in Mass Spec 1				CDs Analyzed in Mass Spec 2
Temperature (°C)	23	24	24	23	23	24	24	23
Humidity (%)	23	52	52	26	22	21	42	21
Sample crystals (%)	100	100	100	100	98.9	100	100	100
Calibrant crystalls (%)	100	100	100	100	100	100	100	100
Isolation efficency Phos-dePhos (%)	93.3	95.5	100	90.9	86.7	93.3	96.2	100

The peptides tested were singly phosphorylated (pTyr) synthetic PDGF-r peptide (1405.6 Da), singly phosphorylated (pThr) CaM kinase II substrate peptide (1437.6 Da), singly phosphorylated (pSer) β-casein peptide (2059.8 Da), and a quadruply phosphorylated (4pSer) β-casein peptide (3121.3 Da). All samples—50 fmol β-casein, 100 fmol CaM kinase II substrate peptide, and 25 fmol PDGF-r peptide—were mixed with 200 fmol BSA tryptic peptides. A sample is considered successfully analyzed when all four phosphopeptides are detected as phosphorylated and as dephosphorylated in the duplicate analysis.