Panel A, left shows a Coomassie Blue–stained 12% polyacrylamide gel after SDS-PAGE analysis of reduced and alkylated samples representing various combinations of 1.5 µM LPL2 and 7 µM GPIHBP1 variants subjected to EDC cross-linking. Lane 2 shows LPL GPIHBP11–131 before cross-linking. Lanes 3–6 show samples after EDC cross-linking: GPIHBP11–131 (lane 3); LPL (lane 4), LPL GPIHBP11–131 (lane 5), and LPL GPIHBP134–131 (lane 6). The covalently bound conjugate representing LPL•GPIHBP11–131 is marked by an asterisk. Right panel shows a Coomassie Blue–stained 4–12% gradient polyacrylamide gel after analysis of EDC cross-linked 3 µM LPL2 alone (lane 8) or in the presence of 15 µM GPIHBP11–33 (lane 9); 15 µM GPIHBP134–131 (lane 10); and 15 µM GPIHBP11–131 (lane 11). The covalent conjugates representing LPL•GPIHBP11–131 and LPL•GPIHBP11–33 are indicated by an asterisk and a solid dot, respectively. Molecular weight markers are shown in lanes 1, 7 & 12. Panel B shows a model of human LPL highlighting the cross-linking sites in GPIHBP1 that were identified by MS (asterisks). Areas that have been assigned as potential interaction sites for GPIHBP1 by HDX-MS are shown in green (for the acidic domain of GPIHBP1) and blue (for the LU domain of GPIHBP1). The position of the interdomain interface in LPL between the NTD and CTD is marked by a dashed line, and three residues within this interface linked to familial chylomicronemia when mutated (S259R, G409R, and E410V) are shown by gray numbers. Basic residues of the heparin-binding site in the catalytic domain of LPL (R279, K280, R282) are shown as sticks. Note, bovine LPL contains two additional residues compared with human LPL, for example Lys296 in human LPL is equivalent to Lys298 in bovine LPL. CTD, C-terminal domain; EDC, N-ethyl-N′-[3-diethylamino)propyl]-carbodiimide; HDX-MS, hydrogen–deuterium exchange mass spectrometry; LPL, lipoprotein lipase; MS, mass spectrometry; NTD, N-terminal domain; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.