Subunit analysis of bovine heart complex I by reversed-phase high-performance liquid chromatography, electrospray ionization-tandem mass spectrometry, and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry

Abstract

An effective method was developed for isolation and analysis of bovine heart complex I subunits. The method uses C18 reversed-phase high-performance liquid chromatography (HPLC) and a water/acetonitrile gradient containing 0.1% trifluoroacetic acid. Employing this system, 36 of the 45 complex I subunits elute in 28 distinct chromatographic peaks. The 9 subunits that do not elute are B14.7, MLRQ, and the 7 mitochondrial-encoded subunits. The method, with ultraviolet (UV) detection, is suitable for either analytical (<50 microg protein) or preparative (>250 microg protein) applications. Subunits eluting in each chromatographic peak were initially determined by matrix-assisted laser desorption/ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS) with subsequent positive identification by reversed-phase HPLC-electrospray ionization (ESI)/tandem mass spectrometry (MS/MS) analysis of tryptic digests. In the latter case, subunits were identified with a 99% probability using Mascot for database searching and Scaffold for assessment of protein identification probabilities. The reversed-phase HPLC subunit analysis method represents a major improvement over previous separation methods with respect to resolution, simplicity, and ease of application.

Figure 1

RP-HPLC elution profile of bovine heart Complex I. Complex I (250 µg) was incubated in 0.1% TFA for 5 min. at RT, and injected onto a C-18 Vydac column (300 Å, 4.6 mm × 250 mm, 10 µm). Subunits were eluted using a complex gradient made from solvent A (H₂O containing 0.1% TFA) and solvent B (CH₃CN containing 0.1% TFA). Elution gradient (dashed line) consisted of: 1) linear gradient from 0% to 30% B in 10 min.; 2) linear gradient from 30% to 42.5% B in 50 min.; 3) linear gradient from 42.5% to 44.5% B in 20 min.; 4) linear gradient from 44.5% to 55% B in 42 min.;5) isocratic elution with 55% B for 5 min.; 6) linear gradient from 55% to 100% B in 36 min.; 7) isocratic elution with 100% B for 5 min.; 8) linear gradient from 100% B to 100% A in 6 min.; and 9) isocratic elution with 100% buffer A for 30 min. to re-equilibrate column for next injection. Elution conditions were 1.0 mL/min. with detection at 214 nm.

Figure 2

Representative MALDI-TOF mass spectra of Complex I subunits that had been isolated by RP-HPLC. Many HPLC elution peaks contain a single subunit, e.g., Panel A: elution peak 2 contains only subunit 9 kDa, m/z = 8452.63; Panel B: HPLC elution peak 3 contains only subunit 15 kDa, m/z = 12571.13; and Panel C: elution peak 23 contains only subunit B14.5b, m/z = 14147 m/z. Some of the other elution peaks contain more than one subunit, e.g., Panel D: HPLC elution peak 16 contains subunit KFYI, m/z = 5824 and subunit AGGG, m/z = 8516. It should be noted that some subunit names, e.g., 9 kDa and 15 kDa, do not accurately reflect the true mass of either subunits.