Analyst Accepted Manuscript This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains. www.rsc.org/analyst Page 1 of 51 Analyst 1 2 3 4 Measuring Masses of Large Biomolecules and Bioparticles 5 6 7 Using Mass Spectrometric Techniques 8 * 9 Wen-Ping Peng , Szu-Wei Chou, Avinash A. Patil 10 11 12 Department of Physics, National Dong Hwa University, Shoufeng, Hualien, 97401, 13 Taiwan, R.O.C. 14 15 16 * To whom correspondence should be addressed, e-mail: [email protected] 17 18 19 Contract/grant sponsor: NSC 102-2112-M-259 -003 -MY3 20 21 Prepared for the Analyst (review article) 22 23 24 25 26 27 Manuscript 28 29 30 31 32 33 34 35 36 37 Accepted 38 39 40 41 42 43 44 45 Analyst 46 47 48 49 50 51 Keywords: large protein complexes, bioparticles, ion source, mass analyzer, detectors, 52 mass measurement, mass assignment, mass resolution 53 54 55 56 57 58 59 60 Analyst Page 2 of 51 1 2 3 4 Abstract 5 6 7 Large biomolecules and bioparticles play a vital role in biology, chemistry, biomedical 8 9 10 science, and physics. Mass is a critical parameter for characterization of large 11 12 13 biomolecules and bioparticles. To achieve mass analysis, choosing a suitable ion 14 15 source is the first step and the instruments of detecting ions, mass analyzers and 16 17 18 detectors, should also be considered. Abundant mass spectrometric techniques have 19 20 21 been proposed to determine the masses of large biomolecules and bioparticles, and 22 23 24 these techniques can be divided into two categories: The first category is to measure 25 26 27 the mass (or size) of intact particles, including single particle quadrupole ion trap Manuscript 28 29 30 mass spectrometry, cell mass spectrometry, charge detection mass spectrometry, and 31 32 33 differential mobility mass analysis; the second category aims to measure the mass and 34 35 36 tandem mass of biomolecular ions, including quadrupole ion trap mass spectrometry, 37 Accepted 38 39 time-of-flight mass spectrometry, quadrupole orthogonal time-of-flight mass 40 41 42 spectrometry and orbitrap mass spectrometry. Moreover, algorithms for mass and 43 44 45 stoichiometry assignment of electrospray mass spectra are developed to obtain Analyst 46 47 accurate structure information and subunit combinations. 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 3 of 51 Analyst 1 2 3 4 1. Introduction 5 6 7 Mass spectrometric techniques can be applied to acquire masses and fragment of 8 9 10 large biomolecules and of bioparticles which facilitate the study of biological process, 11 12 1-3 13 interaction of biomolecules, and their structures and functions. The size of large 14 15 16 biomolecules and bioparticles are in the range from 1 nm to 100 µm. Those large 17 18 biomolecules and bioparticles include protein complexes, viruses, bacteria and cells. 19 20 21 So far, it is still a challenge to detect bioparticles with sizes from 20 nm to 100 nm. 4, 5 22 23 24 Many research groups have tried to develop mass spectrometers to detect the masses 25 26 27 of these sizes. In general, a mass spectrometer includes an ion source, a mass analyzer Manuscript 28 29 30 and a detector and therefore selecting ion sources and designing workable mass 31 32 6 33 analyzers and detectors are a necessity. 34 35 36 Ions are a primary issue for mass analysis. The ion sources used in mass 37 Accepted 38 7 39 spectrometry (MS) for large ions include electrospray ionization (ESI) , 40 41 8, 9 42 matrix-assisted laser desorption/ionization (MALDI) and laser-induced acoustic 43 44 10-12 45 desorption (LIAD) . With ESI ion source, proteins, protein complexes and viral Analyst 46 47 capsids are generated in air and kept intact in vacuum. 13-17 ESI ion source can produce 48 49 50 multiple charged ions with mass-to-charge ratio (m/z) below 100000. However, when 51 52 53 the particle size becomes large, particles gain higher charges on the surfaces (typically 54 55 56 > 50+) and cause highly overlapping ion series, making charge assignment 57 58 59 60 Analyst Page 4 of 51 1 2 3 18-20 4 difficult. By contrast, the MALDI ion source mostly generates singly, doubly 5 6 7 charged peaks which could be easily assigned. Intact ions can be produced by laser 8 9 10 irradiation on matrix crystal. When analyzing large ions, formation of particles is 11 12 13 found in the size range from 10 nm to 1000 nm by irradiating MALDI matrix 14 15 samples. 21, 22 The matrix particles will interfere with viral particles (the size range 16 17 18 from 20 nm to 300 nm) during laser desorption process and thus hinder the analysis of 19 20 21 large bioparticles. Moreover, when the particle mass becomes large (>100 kDa), its 22 23 24 ion velocity is low, making high mass ion detection very challenging.23-25 Besides, 25 26 27 tandem mass analysis of MALDI ions is helpful to acquire the structure information, Manuscript 28 29 30 but it is not well studied yet for m/z > 12000. 26 The LIAD ion source is favorable in 31 32 12, 27, 33 generating particles with the size greater than 50 nm by means of acoustic waves. 34 35 28 36 Multiple pre-charged ions can be generated without matrix interference and fitted to 37 Accepted 38 12, 27 39 single particle analysis with both optical and charge detection. The drawback of 40 41 42 LIAD ion source is low ion generation efficiency. 43 44 45 After ions are generated, they must be transported and guided to the mass Analyst 46 47 analyzers for mass analysis. Typically, by reducing the guiding quadrupole driving 48 49 50 frequency and increasing the buffer gas pressure, high mass ions can be cooled down 51 52 53 and guided to mass analyzers. 29-33 Mass analyzers can be divided into ion trap and 54 55 56 time-of-flight categories. Ion trap mass analyzers such as two dimensional 34-37 and 57 58 59 60 Page 5 of 51 Analyst 1 2 3 38, 39 40 31, 32, 4 three dimensional quadrupole ion trap (QIT) , DC gate ion trap and orbitrap 5 6 41 7 show good trapping efficiency of large particles. Time-of-flight (TOF) mass 8 9 10 analyzers can be run in linear, reflectron, and orthogonal modes and coupled with 11 12 29, 30 13 quadrupole for mass selection, ion activation and isolation. Moreover, no mass 14 15 analyzers can fully cover the whole size ranges of nanoparticles. If the particle density 16 17 18 is known, the particle size can be converted to particle mass. Therefore, differential 19 20 21 mobility analysis (DMA) coupled with electrospray ion source offers an alternative 22 23 24 approach to measure the effective particle sizes and thus characterize large 25 26 27 biomolecules and nanoparticles ranging from a few nanometers to several hundred Manuscript 28 29 30 nanometers.42-44 31 32 33 Detectors are incorporated with mass analyzers to detect ions. The main reason 34 35 36 that the conventional mass spectrometers cannot detect high mass ions is the use of a 37 Accepted 38 39 secondary electron detector, such as an electron multiplier and multi-channel plates 40 41 42 (MCP) which cause low secondary electron yields of high mass ions. New detectors 43 44 27, 40 45, 46 45 including a charge detector , a nanomembrane detector , a secondary ion Analyst 46 47 detector 47, 48 , an active pixel detector 49 , and a cryodetector 50 are therefore designed 48 49 50 and incorporated with mass analyzers to overcome that problem. 46, 47, 51, 52 51 52 53 In general, when determining the masses of large biomolecules and bioparticles, 54 55 56 we could divide mass spectrometric techniques into two categories. The first category 57 58 59 60 Analyst Page 6 of 51 1 2 3 4 aims to measure the masses of intact particles including single particle quadrupole ion 5 6 12, 28, 53 27, 54 7 trap mass spectrometry , cell mass spectrometry , charge detection mass 8 9 40, 55, 56 42, 43 10 spectrometry and differential mobility mass analysis . In single quadrupole 11 12 13 ion trap mass spectrometry and cell mass spectrometry, detectors such as light 14 15 scattering, laser induced fluorescence, a charge detector and an electron multiplier are 16 17 18 developed to overcome the detection limit of large biomolecules and bioparticles. 19 20 21 Charge detection mass spectrometry adopts a non-destructive charge detector to sense 22 23 24 the image charge of particles.