Revealing Higher Order Protein Structure Using Mass Spectrometry

Abstract

The development of rapid, sensitive, and accurate mass spectrometric methods for measuring peptides, proteins, and even intact protein assemblies has made mass spectrometry (MS) an extraordinarily enabling tool for structural biology. Here, we provide a personal perspective of the increasingly useful role that mass spectrometric techniques are exerting during the elucidation of higher order protein structures. Areas covered in this brief perspective include MS as an enabling tool for the high resolution structural biologist, for compositional analysis of endogenous protein complexes, for stoichiometry determination, as well as for integrated approaches for the structural elucidation of protein complexes. We conclude with a vision for the future role of MS-based techniques in the development of a multi-scale molecular microscope. Graphical Abstract ᅟ.

Keywords: Electrospray ionization; Endogenous protein complexes; Folding state in solution; Higher order structure; MALDI; Mass spectrometry; Molecular microscope; Native mass spectrometry; Protein assembly; Protein complex; Protein composition; Stoichiometry; Structural biology; X-ray crystallography.

Figure 1

Hierarchy of protein structures within cells. Mass Spectrometry is playing an increasingly important role in revealing aspects of higher order protein structures as well as the architectures of protein assemblies at all relevant scales within cellular systems. For illustrative purposes, we show a yeast cell nucleus. Embedded in the nuclear envelope at its periphery are nuclear pore complexes (NPCs), the sole mediators of transport into and out of the nucleus. The yeast NPC is an assembly consisting of some 500 protein subunits that are formed through association of a host of protein complexes [2]. One of these, the seven-membered Nup84 protein complex, is shown as an example [3]. There are 16 copies of the Nup84 complex in the yeast NPC [4]

Figure 2

MS helped facilitate several of the obligatory steps that were required during the X-ray structure determination of the ClC chloride transporter [18, 19]. See text for details

Figure 3

Identification of proteins in the highly enriched nuclear pore complex (NPC) fraction [4]. Proteins in the highly enriched NPC fraction were separated by hydroxyapatite HPLC and SDS-PAGE. The number above each lane indicates the corresponding fraction number. Proteins were visualized with Coomassie blue; the approximate molecular mass of proteins as estimated from standards shown on the left side. Bands analyzed by MALDI-TOF mass spectrometry are indicated by the adjacent numbers. The proteins identified in each band are shown in the top panel. Proteins known to directly associate with the NPC are colored blue, whereas proteins believed not to be NPC-associated are colored red. On the top left are listed proteins not found in this separation but identified by MS/MS of reversed phase HPLC-separated peptides (RP/HPLC) or peptide microsequencing (PROT SEQ). Figure adapted from [4] with permission

Figure 4

Protein interactions of the Nup84 complex [41]. (a) Sample of affinity purifications containing Nup84 complex proteins. Affinity-purified protein A (PrA)-tagged proteins and interacting proteins were resolved by SDS-PAGE and visualized with Coomassie blue. The name of the PrA-tagged protein is indicated above each lane. Molecular mass standards (kDa) are indicated to the left of the panel. The bands marked by filled circles at the left of the gel lanes were identified by MS. The identity of the co-purifying proteins is indicated in order below each lane; PrA-tagged proteins are indicated in blue, co-purifying nucleoporins in black, NPC-associated proteins in grey, and other proteins (including contaminants) in red. Each individual gel image was differentially scaled along its length so that its molecular mass standards aligned to a single reference set of molecular mass standards, and contrast-adjusted to improve visibility. (b) The mutual arrangement of the Nup84-complex-associated proteins as visualized by their calculated localization volumes. Figure adapted from [41] with permission

Figure 5

Selection of landmarks in the development of “native MS.” (a) Mass spectra of cytochrome c electrosprayed from variously acidified aqueous solutions. The observed charge distributions reflect the folding state of cytochrome c in the ESI solution [78]; (b) (top panel) early observation of noncovalent ternary complex between the dimeric enzyme HIV-1 protease and a substrate-based inhibitor obtained under native spray conditions [79]; (bottom panel) only the protease monomer was observed when a high amount of collisional energy was injected into the system; (c) m/z spectrum of a mixture of four compounds obtained with a collisional damping interface for an orthogonal injection ESI time-of-flight mass spectrometer [80], showing that it was possible to measure masses ranging from 1000 to 1,000,000 Da in a single mass spectrum; (d) m/z spectrum of the intact rotary ATPase from T. thermophiles, a membrane-embedded molecular machine with mass 659,202 Da [81]. Peaks are assigned to the intact ATPase complex (stars) as well as to losses of the indicated subcomplex and subunits; (e) (left panel) ESI mass spectrum of ~18 MDa Prohead-1 particles from bacteriophage HK97 capsids. A partially resolved series of charge states is observed, allowing the accurate mass calculation indicated [82]; (right panel) charge detection MS, which simultaneously measures the charge and the m/z of individual ions, of bacteriophage P22 procapsid distributions [83]. Shown is the charge versus mass scatter plot for individual P22 procapsid ions centered at mass 23.60 MDa as well as a lower mass cluster of ions centered at 19.84 MDa, attributed to empty capsids. Figures adapted from the indicated references with permission

Figure 6

Native mass spectrometry for determining stoichiometry and subunit connectivity of endogenous protein complexes [89]. (a) Cryomilling, affinity isolation, protease elution, and subsequent native MS analysis of the endogenous Nup84 complex from budding yeast. (b) SDS-PAGE separation and Coomassie staining to assess the post-elution sample handling steps (elution was achieved by cleavage with the HRV 3C protease, later removed by filtration; subsequent buffer exchange was performed with 500 mM ammonium acetate, 0.01% Tween-20). Also shown is the native MS spectrum of the Nup84 complex with (c) low in-source activation and (d) high in-source activation. The structural model for the Nup84 holocomplex, based on integrative structural studies (see text) is also shown. Figure adapted from [89] with permission

Figure 7

Integrated modeling approaches that incorporate MS for the structural elucidation of protein complexes. (a) Chemical crosslinking with MS readout (CX-MS) maps of the Nup84 complex by disuccinimidyl suberate (DSS) crosslinker (top) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) crosslinker (bottom). (b) The Nup84 complex molecular architecture revealed by CX-MS integrative pipeline. (c) Correlation between the number of crosslinks and the accuracy of dimer models as compared to a part of the structure for which a crystallographic dimer structure is available (see text). Accuracy of dimer models as a function of the number and type of crosslinks. Each symbol displays the first and third quartile (lower and upper side of the boxes), the median (red line), as well as minimum and maximum (lower and upper limit of the dashed whiskers, respectively) of the Cα dRMSD with respect to the crystallographic structure for the 100 best-scoring models. (d) (left) Subunit proximities within S. cerevisiae replisome determined by CX-MS. (d) (right) Architecture of the eukaryotic replisome with the proposed DNA path indicated. Red and black lines illustrate possible leading- and lagging-strand DNA. The blue arrow indicates the direction of replisome movement on DNA. The diagram indicates a long path of leading-strand DNA through the entire Mcm ring and then bending back up to Pol ε, requiring about 40 nucleotides of ssDNA. Leading ssDNA is illustrated as going completely through the Mcm2–7 complex and then bending up through the second ‘accessory’ channel of CMG, but this path is speculative. Other DNA paths are possible. Figure adapted from [3, 7] with permission

Figure 8

Vision for a multiscale molecular microscope to define cellular protein assemblies in space and time