Characterization of Phosphorylated Peptides Using Traveling Wave-Based and Drift Cell Ion Mobility Mass Spectrometry

100

Structural separations on the basis of gas-phase ion mobility-mass spectrometry are increasingly used for the analysis of complex biological samples. As a tool to elucidate biomolecular structure, ion mobility-mass spectrometry methods are unique in that direct molecular structural information is obtained for all resolved species, largely irrespective of the complexity of the sample. Computational approaches are used to interpret and discern structural details consistent with the empirical results. To a first approximation, correlations of mobility with mass allow for qualitative identification of the molecular class to which a particular species belongs. These correlations allow simultaneous characterization of different classes of biomolecules, which provides a means for combining omics measurements, such as lipidomics, proteomics, glycomics, and metabolomics, in the same analysis. Examination of the correlation of fine structure reveals that specific structural motifs, chemical functionality, chemical connectivity, and composition may also be determined, depending on the specific biomolecular class. Mapping the coarse and fine structure in ion mobility-mass spectrometry conformation space measurements provides an atlas for interpretation and discovery in complicated spectra. as-phase separations on the basis of migration and diffusion of ions through a neutral gas under the influence of an applied field have existed for over a century. The first examples of gasphase ion mobility (IM) include the quantitative studies of ionized gases by Rutherford shortly after the discovery of X-rays [1,2]. To place these early IM experiments in the context of mass spectrometry (MS), it would be almost another decade before J. J. Thomson would construct his first mass spectrograph [3]. Over the historic span of IMS development, the technique has been referred to by several names, including plasma chromatography [4], ion chromatography [5], and the currently accepted term ion mobility spectrometry (IMS). A detailed account on the historic development of IMS and milestones in the progress of gas-phase IM techniques is described elsewhere [6,7]. For many years, IMS separations were used for the fundamental study of atomic and small molecular ions in plasma physics. In the 1960s and 70s, a transition occurred from using IMS primarily as a fundamental physics research tool to using it as a separations device for analytical and physical chemistry applications. Notably, the early 1960s also marked the first reports of combining IMS with MS (IM-MS) [8,9].On the basis of these early IM-MS instruments, it was proposed that with proper calibration, IMS instruments could be operated as atmospheric pressure mass spectrometers in that mass could be assigned to a particular IM drift time [10,11]. This supposition was derived from plotting curves of mass as a function of mobility, which resulted in highly correlated calibration curves. However, this notion was qualified by Horning and colleagues in that highly correlated mass-mobility relat...

Section: Structural Separations In Conformation Space-a Correlation Fmentioning

confidence: 99%

The mass-mobility correlation redux: The conformational landscape of anhydrous biomolecules

McLean

2009

100

“…Ion mobility arrival time distributions were converted to collision cross sections via calibration against the collision cross sections of myoglobin, cytochrome c and ubiquitin as published previously [20,21]. Briefly, the arrival time of each peak was first measured in scans.…”

Section: Collision Cross Section Measurementsmentioning

confidence: 99%

Methodology for measuring conformation of solvent-disrupted protein subunits using T-WAVE ion mobility MS: An investigation into eukaryotic initiation factors

Leary

Schenauer

Ştefănescu

et al. 2009

Self Cite

The methodology developed in the research presented herein makes use of chaotropic solvents to gently dissociate subunits from an intact macromolecular complex and subsequently allows for the measurement of collision cross section (CCS) for both the recombinant (R-eIF3k) and solvent dissociated form of the subunit (S-eIF3k). In this particular case, the k subunit from the eukaryotic initiation factor 3 (eIF3) was investigated in detail. Experimental and theoretical CCS values show both the recombinant and solvent disrupted forms of the protein to be essentially the same. The ultimate goal of the project is to structurally characterize all the binding partners of eIF3, determine which subunits interact directly, and investigate how subunits may change conformation when they form complexes with other proteins.

“…In brief, normalized cross sections (corrected for charge and reduced mass) were plotted against corrected arrival times (corrected to exclude time spent outside the ion mobility cell) to create a calibration with a power-series fit. The calibration allows one to estimate the cross section of a molecule of interest provided that the mobilities (corrected arrival times) for that molecule lie within the mobilities observed for the calibrant used, irrespective of the size range of cross sections for the calibrant [34,35]. The calibration was used to estimate rotationally averaged collision cross sections of hemoglobin monomer, dimer, and tetramer for the different charge states observed, based on their arrival time distributions, provided that their corrected arrival times fell along the calibration curve.…”

Section: Calibration Modeling and Estimation Of Cross Sectionmentioning

confidence: 99%

Probing hemoglobin structure by means of traveling-wave ion mobility mass spectrometry

Scarff

Patel

Thalassinos

et al. 2008

Self Cite

Hemoglobin (Hb) is a tetrameric noncovalent complex consisting of two ␣-and two ␣ ␤-globin ␤ chains each associated with a heme group. Its exact assembly pathway is a matter of debate. Disorders of hemoglobin are the most common inherited disorders and subsequently the molecule has been extensively studied. This work attempts to further elucidate the structural properties of the hemoglobin tetramer and its components. Gas-phase conformations of hemoglobin tetramers and their constituents were investigated by means of traveling-wave ion mobility mass spectrometry. Sickle (HbS) and normal (HbA) hemoglobin molecules were analyzed to determine whether conformational differences in their quaternary structure could be observed. Rotationally averaged collision cross sections were estimated for tetramer, dimer, apo-, and holo-monomers with reference to a protein standard with known cross sections. Estimates of cross section obtained for the tetramers were compared to values calculated from X-ray crystallographic structures. HbS was consistently estimated to have a larger cross section than that of HbA, comparable with values obtained from X-ray crystallographic structures. Nontetrameric species observed included apo-and holo-forms of ␣-and ␣ ␤-monomers and ␤ heterodimers; ␣-and ␣ ␤-monomers in both apo-and holo-forms were found to have similar ␤ cross sections, suggesting they maintain a similar fold in the gas phase in both the presence and the absence of heme. Heme-deficient dimer, observed in the spectrum when analyzing commercially prepared Hb, was not observed when analyzing fresh blood. This implies that holo-␣-apo-␣ ␤ is not an essential intermediate within the The typical electrospray ionization (ESI) mass spectrum of a protein consists of an envelope of peaks attributed to a series of multiply charged gas-phase ions that can indicate the stability and compactness of its structure in the gas phase [4,5]. Multiply charged ions are produced by proton attachment, predominantly to exposed basic sites on the protein [6], and those of lowest charge are thought to be most representative of native structure [7]. A highly compact protein would have fewer exposed basic sites than those of an unfolded conformation of the same protein and thus would accept fewer charges [4,8].Ion mobility spectrometry is a shape-selective technique, based on the time taken for an ion to traverse a mobility cell containing an inert gas under the influence of a weak electric field [9]. This time is related to the rotationally averaged collision cross section, mass, and charge of the ion. The coupling of ion mobility separation with mass spectrometry has provided a powerful method for the analysis of complex mixtures and for the determination of molecular structure [10].Ion mobility mass spectrometry has emerged as a complementary technique to the well-established methods of X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy for three-dimensional protein structure analysis [11]. There is now substantial evidence to support...