Figure 2.

Catalytic β subunit expression in different human cell lines. A, RT-PCR products specific for PSMB11 (β5t subunit) or actin (as control) obtained from different immortalized or tumor-derived human cell lines. The PSMB11-specific band is marked with a red arrow. Specificity has been confirmed by cloning and sequencing the cDNA extracted from the band. B, MS identification of the β5t subunit in the cell lysate of the same cell lines shown in A. Cell lysates have been separated on an SDS gel and stained with Coomassie (left panel; only some representative cell samples are shown), the proteins framed in the picture have been cut and digested by trypsin, and the β5t subunit–specific peptides have been detected by MS. Only for the C5.5 cell line have we identified the β5t subunit products (marked with bold letters; 19 specific peptides depicted below the gel) with significant MS/MS spectra (the spectrum of one of them is depicted in the right panel). C, Western blot assays for proteasome catalytic subunits, which have been carried out after separation of 0.5 μg of purified 20S proteasomes by SDS-PAGE, are shown. D, relative quantification of the subunits β5 (PSB5_human) and β5t (PSB11_human) of purified 20S proteasome after absolute quantification with AQUA peptides. Shown are representative MALDI mass spectra of the tryptic peptide ²²⁶DAYSGGAVNLYHVR²³⁹ with [M + H]⁺_exp = 1521.78 and its heavy analogue with [M + H]⁺_exp = 1528.80 of β5 subunit at spot 42 and ²¹⁶DAYSGGSVDLFHVR²²⁹ with [M + H]⁺_exp = 1522.77 and its heavy analogue with [M + H]⁺_exp = 1529.79 of β5t subunit at spot 69. The calculation of the absolute amount of the tryptic peptides is performed by comparison of the MS peak areas with those of the corresponding AQUA peptides.