© 2012 Nature America, Inc. All rights reserved. BRIEF COMMUNICATIONS NATURE METHODS | ADVANCE ONLINE PUBLICATION | 1 peptides, we show here that these instruments can be modified to analyze very large native protein assemblies. We made dedicated instrumental modifications to an Exactive Plus instrument (ThermoFisher Scientific) with a higher-energy collision-induced dissociation (HCD) option (Supplementary Fig. 1). Briefly, this included altering software to allow detection of ions at higher m/z range, tuning radiofrequency voltages applied to transport multipoles and altering the pressure in the HCD cell (Online Methods). For introduction of samples, we used a static nanospray source with gold-plated capillaries made in-house. Initially, to probe the boundaries of m/z detection and mass resolution of the modified instrument, we analyzed the salt CsI, which forms clusters of increasing molecular weight and is often used as a mass calibrant. We detected CsI clusters up to m/z of 18,000 (Supplementary Fig. 2). For comparison, we also ran CsI High-sensitivity Orbitrap mass analysis of intact macromolecular assemblies Rebecca J Rose 1,2 , Eugen Damoc 3 , Eduard Denisov 3 , Alexander Makarov 3 & Albert J R Heck 1,2 The analysis of intact protein assemblies in native-like states by mass spectrometry offers a wealth of information on their biochemical and biophysical properties. Here we show that the Orbitrap mass analyzer can be used to measure protein assemblies of molecular weights approaching one megadalton with sensitivity down to the detection of single ions. Minor instrumental modifications enabled the measurement of various protein assemblies with outstanding mass-spectral resolution. The analysis of intact protein assemblies by mass spectrometry can provide essential information for understanding various biological and biophysical properties, from protein identity to sample heterogeneity, ligand binding and substrate turnover to structural topology and dynamics of assembly 1,2 . These ‘native mass spectrometry’ experiments have been performed on various biological systems over recent years and have advanced such that megadalton complexes can now be analyzed 3 . To observe these high-molecular-weight ions, commercially available instruments are typically modified in-house 4,5 . These analyses to date have been virtually exclusively the domain of time-of-flight (TOF) mass analyzers, owing to the capacity of these instruments to access very high mass-to-charge ratio (m/z) values (up to 100,000 Th) 6 . Enhanced pressures and altered electronics (for example, quadrupole frequency, collision energy and TOF pusher fre- quency), as well as specialized detectors, have been developed to improve transmission and measurement of very large ions 4,5,7 . The demand for mass spectrometry–based studies on large biological molecules is increasing, especially in the area of struc- tural biology, but also for applications within, for example, the biopharmaceutical industry 8 , necessitating varied approaches and improved technologies. Here we report the use of the Orbitrap mass analyzer for measuring intact protein assemblies with molec- ular weights approaching one million daltons. Since the introduc- tion of the first Orbitrap-based mass spectrometers in 2005, this mass analyzer has become increasingly popular 9 . Although these instruments are typically used to analyze small molecules and 1 Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute of Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands. 2 Netherlands Proteomics Center, Utrecht, The Netherlands. 3 Thermo Fisher Scientific (Bremen), Bremen, Germany. Correspondence should be addressed to A.J.R.H. (a.j.r.heck@uu.nl). RECEIVED 20 JUNE; ACCEPTED 17 SEPTEMBER; PUBLISHED ONLINE 14 OCTOBER 2012; DOI:10.1038/NMETH.2208 5,000 0 7,500 10,000 12,500 5,840 5,870 25+ m/z m/z 149 kDa a Intensity Intensity 5,000 7,500 10,000 12,500 m/z 210 kDa and 253 kDa b 0 Intensity 5,000 7,500 10,000 730 kDa c 0 Intensity 12,500 m/z 801 kDa d 0 Intensity 5,000 7,500 10,000 12,500 m/z Figure 1 | Orbitrap-based mass spectra of intact proteins and protein assemblies. (a–d) Native mass spectra of IgG antibody (a), bacteriophage HK97 capsid pentamers and hexamers (b), yeast 20S proteasome (c) and E. coli GroEL (d). Illustrative crystal structures are shown for each protein. Inset in a shows an enlargement of the 25+ charge state of IgG1.