Trapped Ion Mobility Spectrometry of Native Macromolecular Assemblies

Abstract

The structural elucidation of native macromolecular assemblies has been a subject of considerable interest in native mass spectrometry (MS), and more recently in tandem with ion mobility spectrometry (IMS-MS), for a better understanding of their biochemical and biophysical functions. In the present work, we describe a new generation trapped ion mobility spectrometer (TIMS), with extended mobility range (K 0 = 0.185−1.84 cm 2 •V −1 •s −1 ), capable of trapping high-molecular-weight (MW) macromolecular assemblies. This compact 4 cm long TIMS analyzer utilizes a convex electrode, quadrupolar geometry with increased pseudopotential penetration in the radial dimension, extending the mobility trapping to high-MW species under native state (i.e., lower charge states). The TIMS capabilities to perform variable scan rate (Sr) mobility measurements over short time (100−500 ms), high-mobility resolution, and ion-neutral collision cross-section (CCS N 2 ) measurements are presented. The trapping capabilities of the convex electrode TIMS geometry and ease of operation over a wide gas flow, rf range, and electric field trapping range are illustrated for the first time using a comprehensive list of standards varying from CsI clusters (n = 6−73), Tuning Mix oligomers (n = 1−5), common proteins (e.g., ubiquitin, cytochrome C, lysozyme, concanavalin (n = 1−4), carbonic anhydrase, β clamp (n = 1−4), topoisomerase IB, bovine serum albumin (n = 1−3), topoisomerase IA, alcohol dehydrogenase), IgG antibody (e.g., avastin), protein−DNA complexes, and macromolecular assemblies (e.g., GroEL and RNA polymerase (n = 1−2)) covering a wide mass (up to m/z 19 000) and CCS range (up to 22 000 Å 2 with <0.6% relative standard deviation (RSD)).

Tandem‐trapped ion mobility spectrometry/mass spectrometry coupled with ultraviolet photodissociation

“…Indeed, the mobility separation in TIMS‐2 is capable of separating the various fragment ions to such an extent that their isotope patterns are essentially baseline resolved. We stress two facts: (a) our mobility analysis is carried out at a scan rate of ~1.9 V ms −1 which is about 5–10 times higher than what is typically employed 39–42,69 . This means that there is ample opportunity to improve the mobility separation of the ions shown in Figure 7 by reducing the mobility scan rate, thereby improving the separation of fragment ions; (b) the data indicate the presence of fragment ion species that differ in the presence of one or two hydrogen atoms; and (c) the instrument discussed here enables PASEF 44 workflows in which the fragment ions produced from UVPD are isolated and subsequently fragmented in the quadrupole/collision cell.…”

“…We stress two facts: (a) our mobility analysis is carried out at a scan rate of $1.9 V ms À1 which is about 5-10 times higher than what is typically employed. [39][40][41][42]69 This means that there is ample opportunity to improve the mobility separation of the ions shown in Figure 7 demonstrated utility for protein top-down analysis. Therefore, we anticipate that the instrument described here will prove particularly valuable for structural proteomics studies because it enables PASEFassisted top-down analysis of proteins and protein complexes.…”

Structural O-Glycoform Heterogeneity of the SARS-CoV-2 Spike Protein Receptor-Binding Domain Revealed by Top-Down Mass Spectrometry

“…TIMS is a relatively recent high-resolution ion mobility method in which multiple ions are held in place against a moving gas under an electric field and then ejected at characteristic elution voltages from the TIMS cell enabling the determination of ion mobility (1/ K 0 ) for structural analysis. 53 − 55 The mobility measured in TIMS allows collision cross-section (CCS) determination of protein conformers, which relates to their overall size and shape, and consequently can be used to evaluate changes in the three-dimensional structure. 54 , 57 However, ion mobility mapping and MS 1 analysis reveal enormous complexity in the native S-RBD sample ( Figure S2 ).…”

Trapped Ion Mobility Spectrometry and Parallel Accumulation–Serial Fragmentation in Proteomics

“…As TIMS and PASEF are entering routine use, ongoing developments in both hard-and software are further expanding its capabilities. Recently, Fernandez-Lima and co-workers introduced a new TIMS design with convex electrodes that generates a higher pseudopotential and thereby extends the analytically accessible mass and mobility range up to macromolecular assemblies such as RNA polymerase or the 801 kDa GroEL complex 109 . As mentioned above, using a brighter ion source and a TIMS design with increased charge capacity, we developed an ultra-high sensitivity workflow for label-free quantitative proteomics of single human cancer cells 73 .…”