Top-down Proteoform Analysis by LC-MS

LC-MS analysis was carried out using a NanoAcquity ultra-high pressure LC system (Waters) coupled to a high-resolution Impact II quadrupole time-of-flight mass spectrometer (Bruker Daltonics) (67 (link)). Five hundred nanograms of total sarcomeric protein (per sample) were separated using a home-packed PLRP column (PLRP-S, 1,000 Å pore size, 10-µm particle size, 500-µm inner diameter) using an organic gradient of 5 to 95% mobile phase B (mobile phase A: 0.1% formic acid in water; mobile phase B: 0.1% formic acid in 50:50 acetonitrile:ethanol) at a constant flow rate of 14 µL/min. Individual protein fractions were collected following reversed-phase LC separation for ultrahigh-resolution top-down MS using a 7-T linear ion trap (LTQ)/FTICR mass spectrometer (Thermo Scientific) or 12-T solariX FTICR mass spectrometer (Bruker Daltonics) equipped with an automated chip-based nano electrospray ionization source (Triversa NanoMate; Advion Bioscience). Proteoforms of interest were first isolated in the gas phase and fragmented by ECD and CAD.

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Tucholski T., Cai W., Gregorich Z.R., Bayne E.F., Mitchell S.D., McIlwain S.J., de Lange W.J., Wrobbel M., Karp H., Hite Z., Vikhorev P.G., Marston S.B., Lal S., Li A., dos Remedios C., Kohmoto T., Hermsen J., Ralphe J.C., Kamp T.J., Moss R.L, & Ge Y. (2020). Distinct hypertrophic cardiomyopathy genotypes result in convergent sarcomeric proteoform profiles revealed by top-down proteomics. Proceedings of the National Academy of Sciences of the United States of America, 117(40), 24691-24700.

Publication 2020

NanoAcquity ultra-high pressure LC system Impact II quadrupole time-of-flight mass spectrometer 12-T solariX FTICR mass spectrometer Triversa NanoMate

Acetonitrile Chip Ethanol Formic acid Pressure Protein Sarcomeric

Corresponding Organization :

Other organizations : University of Wisconsin–Madison, University of Wisconsin Carbone Cancer Center, Imperial College London, University of Sydney, La Trobe University, Victor Chang Cardiac Research Institute

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Variable analysis

independent variables

Organic gradient of 5 to 95% mobile phase B (mobile phase A: 0.1% formic acid in water; mobile phase B: 0.1% formic acid in 50:50 acetonitrile:ethanol)

dependent variables

Proteoforms of interest

control variables

Constant flow rate of 14 µL/min

controls

No positive or negative controls were explicitly mentioned.

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