TABLE 1.
Determination of ANGPTL3/8 and ANGPTL4/8 protein ratios by mass spectrometry
| Sample | Molar Ratio of ANGPTL3 Compared with Labeled Peptides | Molar Ratio of ANGPTL4 Compared with Labeled Peptides | Molar Ratio of ANGPTL8 Compared with Labeled Peptides | Protein Ratio for Each Respective Complex | |||||||
| Peptide #1 | Peptide #2 | Average | Peptide #1 | Peptide #2 | Average | Peptide #1 | Peptide #2 | Average | ANGPTL3/8 | ANGPTL4/8 | |
| Recombinant complexes | |||||||||||
| ANGPTL3/8 | |||||||||||
| Sample #1 | 9.60 | 12.96 | 11.28 | — | — | — | 3.64 | 2.92 | 3.28 | 3.4 | — |
| Sample #2 | 9.19 | 13.74 | 11.47 | — | — | — | 3.64 | 3.04 | 3.34 | 3.4 | — |
| Sample #3 | 8.06 | 13.45 | 10.75 | — | — | — | 3.23 | 2.92 | 3.08 | 3.5 | — |
| SD | 0.80 | 0.40 | 0.37 | — | — | — | 0.24 | 0.07 | 0.14 | 0.03 | — |
| ANGPTL4/8 | |||||||||||
| Sample #1 | — | — | — | 0.71 | 0.81 | 0.76 | 0.88 | 0.74 | 0.81 | — | 0.9 |
| Sample #2 | — | — | — | 0.54 | 0.67 | 0.60 | 0.68 | 0.57 | 0.62 | — | 1.0 |
| Sample #3 | — | — | — | 0.68 | 0.92 | 0.80 | 0.74 | 0.62 | 0.68 | — | 1.2 |
| SD | — | — | — | 0.09 | 0.13 | 0.11 | 0.11 | 0.09 | 0.10 | — | 0.13 |
| Endogenous complexes | |||||||||||
| ANGPTL3/8 | |||||||||||
| Sample #1 | 1.27 | 1.34 | 1.31 | — | — | — | 0.37 | 0.38 | 0.37 | 3.5 | — |
| Sample #2 | 1.25 | 1.27 | 1.26 | — | — | — | 0.37 | 0.36 | 0.36 | 3.4 | — |
| SD | 0.02 | 0.05 | 0.03 | — | — | — | 0.004 | 0.01 | 0.01 | 0.08 | — |
| ANGPTL4/8 | |||||||||||
| Sample #1 | — | — | — | 0.041 | 0.055 | 0.048 | 0.062 | 0.058 | 0.060 | — | 0.8 |
| Sample #2 | — | — | — | 0.043 | 0.052 | 0.048 | 0.059 | 0.062 | 0.061 | — | 0.8 |
| SD | — | — | — | 0.002 | 0.002 | 0.00 | 0.002 | 0.003 | 0.01 | — | 0.01 |
ANGPTL3/8 and ANGPTL4/8 complexes were digested using trypsin and Lys-C. Identical molar amounts of SIL peptides were spiked into samples during digestion, and the ratio of unlabeled to labeled peptides was determined. Stoichiometries of protein complexes were determined by comparing the averaged ratios derived from two peptides per protein. Data for recombinant complexes were derived from a single preparation performed in triplicate. Data for endogenous complexes were derived from a serum pool from 20 healthy donors and performed in duplicate. SDs are shown for each peptide in the technical replicates and for the protein ratios in the respective complexes.