Figure 1.
Comparison of the abilities of four different detergents to extract membrane proteins.
(A) SDS-PAGE analysis of the proteins in the supernatants of PM-enriched fraction dissolved by different detergents followed by centrifugation; (B) SDS-PAGE analysis of the proteins remaining in the pellets after detergent extraction. For SDS-PAGE analysis, the pellets were dissolved by loading buffer containing 4% SDS.
Figure 2.
Enzyme activity measured in the presence of different concentrations of SL.
(A) Trypsin. BAEE was hydrolyzed by trypsin to UV-absorbing product BA. Trypsin activity was measured as a slope of the UV absorbance change at A253 nm. (B) Chymotrypsin. BTEE was hydrolyzed by chymotrypsin to UV-absorbing product BT. Chymotrypsin activity was measured as a slope of the UV absorbance change at A256 nm. Data were averaged from three measurements.
Figure 3.
MALDI-TOF MS spectra of tryptic digests of BSA.
(A) mixed with matrix (1∶1) without addition of SL; (B and C) mixed with matrix (1∶1) after addition of SL (the final concentrations were 0.1% and 0.5%, respectively); (D–F) mixed with matrix (1∶1) after addition of SL (the final concentrations were 0.1%, 0.5% and 1.0%, respectively) and then removing it by acidification and phase-transfer method.
Table 1.
Comparative analysis of standard protein mixture identified with CapLC-MS/MS after digested by four different strategies.
Figure 4.
Comparison of the distributions of membrane proteins based on their function annotations identified by the three different detergent-assisted methods.
The merged results from triplicate analysis in each method were used for comparison.
Table 2.
Comparative analysis of proteins and their matching peptides identified by three strategies from rat liver plasma membrane-enriched fraction.
Figure 5.
Comparison of the transmembrane proteins identified from rat liver PM-enriched fraction in three different detergent-assisted methods.
(A) Comparison of the distributions of all the identified integral membrane proteins as a function of TMDs. The data merged from triplicate analysis were used for comparison. (B) Comparison of sequence coverages of the integral membrane protein isoform GLAST-1 of excitatory amino acid transporter 1 (IPI00324377) identified by RGS, SDC or SL method. The sequence coverages were visualized with the TOPO2 transmembrane protein graphics program. The tryptic peptides identified by all three methods were in red, and those newly identified only by SL method were in blue, whose two tryptic cleavage sites inside the TMDs are indicated with black arrows.
Figure 6.
Comparison of the distributions of proteins and their matching peptides identified based on the three different digestion methods.
(A) Distribution of identified proteins as a function of the calculated grand average of hydropathy (GRAVY) values. (B) Distribution of identified peptides as a function of the calculated grand average of hydropathy (GRAVY) values. The data merged from triplicate analysis were used for comparison.