Ion Mobility Mass Spectrometry of Peptide Ions: Effects of Drift Gas and Calibration Strategies

One difficulty in using ion mobility (IM) mass spectrometry (MS) to improve the specificity of peptide ion assignments is that IM separations are performed using a range of pressures, gas compositions, temperatures, and modes of separation, which makes it challenging to rapidly extract accurate shape parameters. We report collision cross section values (Ω) in both He and N₂ gases for 113 peptide ions determined directly from drift times measured in a low-pressure, ambient temperature drift cell with radio-frequency (rf) ion confinement. These peptide ions have masses ranging from 231 to 2969 Da, Ω_He of 89–616 Ų, and Ω_N₂ of 151–801 Ų; thus, they are ideal for calibrating results from proteomics experiments. These results were used to quantify the errors associated with traveling-wave Ω measurements of peptide ions and the errors concomitant with using drift times measured in N₂ gas to estimate Ω_He. More broadly, these results enable the rapid and accurate determination of calibrated Ω for peptide ions, which could be used as an additional parameter to increase the specificity of assignments in proteomics experiments

Intrinsic disorder in proteins: a challenge for (un)structural biology met by ion mobility-mass spectrometry

The link between structure and function of a given protein is a principal tenet of biology. The established approach to understand the function of a protein is to ‘solve’ its structure and subsequently investigate interactions between the protein and its binding partners. However, structure determination via crystallography or NMR is challenging for proteins where localized regions or even their entire structure fail to fold into a three-dimensional form. These so called IDPs (intrinsically disordered proteins) or intrinsically disordered regions constitute up to 40% of all expressed proteins, and a much higher percentage in proteins involved in the proliferation of cancer. For these proteins, there is a need to develop new methods for structural characterization which exploit their biophysical properties. IM (ion mobility)–MS is uniquely able to examine both absolute conformation(s), populations of conformation and also conformational change, and is therefore highly applicable to the study of IDPs. The present article details the technique of IM–MS and illustrates its use in assessing the relative disorder of the wild-type p53 DNA-core-binding domain of cellular tumour antigen p53. The IM data were acquired on a Waters Synapt HDMS instrument following nESI (nanoelectrospray ionization) from ‘native’ and low-pH solution conditions.</jats:p

N-linked glycosylation of native and recombinant cauliflower xyloglucan endotransglycosylase 16A.

The gene encoding a XET (xyloglucan endotransglycosylase) from cauliflower ( Brassica oleracea var. botrytis ) florets has been cloned and sequenced. Sequence analysis indicated a high degree of similarity to other XET enzymes belonging to glycosyl hydrolase family 16 (GH16). In addition to the conserved GH16 catalytic sequence motif EIDFE, there exists one potential N-linked glycosylation site, which is also highly conserved in XET enzymes from this family. Purification of the corresponding protein from extracts of cauliflower florets allowed the fractionation of a single, pure glycoform, which was analysed by MS techniques. Accurate protein mass determination following the enzymic deglycosylation of this glycoform indicated the presence of a high-mannose-type glycan of the general structure GlcNAc2Man6. LC/MS and MS/MS (tandem MS) analysis provided supporting evidence for this structure and confirmed that the glycosylation site (underlined) was situated close to the predicted catalytic residues in the conserved sequence YLSSTNNEHDEIDFEFLGNRTGQPVILQTNVFTGGK. Heterologous expression in Pichia pastoris produced a range of protein glycoforms, which were, on average, more highly mannosylated than the purified native enzyme. This difference in glycosylation did not influence the apparent enzymic activity of the enzyme significantly. However, the removal of high-mannose glycosylation in recombinant cauliflower XET by endoglycosidase H, quantified by electrospray-ionization MS, caused a 40% decrease in the transglycosylation activity of the enzyme. No hydrolytic activity was detected in native or heterologously expressed BobXET16A, even when almost completely deglycosylated

Effects of Drift Gas on Collision Cross Sections of a Protein Standard in Linear Drift Tube and Traveling Wave Ion Mobility Mass Spectrometry

There has been a significant increase in the use of ion mobility mass spectrometry (IM-MS) to investigate conformations of proteins and protein complexes following electrospray ionization. Investigations which employ traveling wave ion mobility mass spectrometry (TW IM-MS) instrumentation rely on the use of calibrants to convert the arrival times of ions to collision cross sections (CCS) providing “hard numbers” of use to structural biology. It is common to use nitrogen as the buffer gas in TW IM-MS instruments and to calibrate by extrapolating from CCS measured in helium via drift tube (DT) IM-MS. In this work, both DT and TW IM-MS instruments are used to investigate the effects of different drift gases (helium, neon, nitrogen, and argon) on the transport of multiply charged ions of the protein myoglobin, frequently used as a standard in TW IM-MS studies. Irrespective of the drift gas used, recorded mass spectra are found to be highly similar. In contrast, the recorded arrival time distributions and the derived CCS differ greatly. At low charge states (7 ≤ <i>z</i> ≤ 11) where the protein is compact, the CCS scale with the polarizability of the gas; this is also the case for higher charge states (12 ≤ <i>z</i> ≤ 22) where the protein is more unfolded for the heavy gases (neon, argon, and nitrogen) but not the case for helium. This is here interpreted as a different conformational landscape being sampled by the lighter gas and potentially attributable to increased field heating by helium. Under nanoelectrospray ionization (nESI) conditions, where myoglobin is sprayed from an aqueous solution buffered to pH 6.8 with 20 mM ammonium acetate, in the DT IM-MS instrument, each buffer gas can yield a different arrival time distribution (ATD) for any given charge state

Differentiation of Aspartic and Isoaspartic Acid Using 193 nm Ultraviolet Photodissociation Mass Spectrometry

Spontaneous conversion of aspartic acid (Asp) to isoaspartic acid (isoAsp) is a ubiquitous modification that influences the structure and function of proteins. This modification of Asp impacts the stability of biotherapeutics and has been linked to the development of neurodegenerative diseases. We explored the use of 193 nm ultraviolet photodissociation (UVPD) to distinguish Asp and isoAsp in the protonated and deprotonated peptides. The differences in the relative abundances of several fragment ions uniquely generated by UVPD were used to differentiate isomeric peptide standards containing Asp or isoAsp. These fragment ions result from the cleavage of bonds N-terminal to Asp/isoAsp residues in addition to the side-chain losses from Asp/isoAsp or the losses of COOH, CO2, CO, or H2O from y-ions. Fragmentation of Asp-containing tryptic peptides using UVPD resulted in more enhanced w/w + 1/y – 1/x ions, while isoAsp-containing peptides yielded more enhanced y – 18/y – 45/y – 46 ions. UVPD was also used to identify an isomerized peptide from a tryptic digest of a monoclonal antibody. Moreover, UVPD of a protonated nontryptic peptide resulted in more enhanced y ions N- and C-terminal to isoAsp and differences in b/y ion ratios that were used to identify the isoAsp peptide

Native-MS Analysis of Monoclonal Antibody Conjugates by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Antibody-drug conjugates (ADCs) are an important class of therapeutic molecule currently being used to treat HER2-positive metastatic breast cancer, relapsed or refractory Hodgkin lymphoma, systemic anaplastic large cell lymphoma, relapsed or refractory B-cell precursor acute lymphoblastic leukemia, and acute myeloid leukemia. An ADC typically consists of a small molecule or peptide-based cytotoxic moiety covalently linked, via lysine or cysteine residues, to a monoclonal antibody (mAb) scaffold. Mass spectrometric (MS) characterization of these molecules affords highly accurate molecular weight (MW) and drug-to-antibody ratio (DAR) determination and is typically performed using orthogonal acceleration time-of-flight (oa-ToF) analyzers and more recently, Orbitrap instruments. Herein we describe for the first time the use of a 15 T solariX Fourier transform ion cyclotron mass spectrometer to characterize an IgG1 mAb molecule conjugated with biotin via native lysine and cysteine residues, under native-MS and solution conditions. The cysteine–biotin conjugates remained fully intact, demonstrating the ability of the FT-ICR to maintain the noncovalent interactions and efficiently transmit labile protein complexes. Native-MS was acquired and is displayed in magnitude mode using a symmetric Hann apodization function. Baseline separation is achieved on all covalent biotin additions, for each charge state, for both the lysine– and cysteine–biotin conjugates. Average DAR values obtained by native-MS for the lysine conjugate are compared to those derived by denaturing reversed phase liquid chromatography using an oa-ToF MS system (1.56 ± 0.02 versus 2.24 ± 0.02 for the 5 equivalent and 3.99 ± 0.09 versus 4.43 ± 0.01 for the 10 equivalent, respectively). Increased DAR value accuracy can be obtained for the higher biotin-load when using standard ESI conditions as opposed to nanoESI native-MS conditions

Characterizing the Size and Composition of Saposin A Lipoprotein Picodiscs

Saposin A (SapA) lipoprotein discs, also known as picodiscs (PDs), represent an attractive method to solubilize glycolipids for protein interaction studies in aqueous solution. Recent electrospray ionization mass spectrometry (ESI-MS) data suggest that the size and composition of 1-palmitoyl-2-oleoyl-<i>sn</i>-glycero-3-phosphocholine (POPC)-containing PDs at neutral pH differs from those of <i>N</i>,<i>N</i>-dimethyldodecylamine <i>N</i>-oxide determined by X-ray crystallography. Using high-resolution ESI-MS, multiangle laser light scattering (MALLS), and molecular dynamics (MD) simulations, the composition, heterogeneity, and structure of POPC–PDs in aqueous ammonium acetate solutions at pH 4.8 and 6.8 were investigated. The ESI-MS and MALLS data revealed that POPC–PDs consist predominantly of (SapA dimer + <i>i</i>POPC) complexes, with <i>i</i> = 23–29, and have an average molecular weight (MW) of 38.2 ± 3.3 kDa at pH 4.8. In contrast, in freshly prepared solutions at pH 6.8, POPC–PDs are composed predominantly of (SapA tetramer + <i>i</i>POPC) complexes, with <i>i</i> = 37–60, with an average MW of 68.0 ± 2.7 kDa. However, the (SapA tetramer + <i>i</i>POPC) complexes are unstable at neutral pH and convert, over a period of hours, to (SapA trimer + <i>i</i>POPC) complexes, with <i>i</i> = 29–36, with an average MW of 51.1 ± 2.9 kDa. The results of molecular modeling suggest spheroidal structures for the (SapA dimer + <i>i</i>POPC), (SapA trimer + <i>i</i>POPC), and (SapA tetramer + <i>i</i>POPC) complexes in solution. Comparison of measured collision cross sections (Ω) with values calculated for gaseous (SapA dimer + 26POPC)⁸⁺, (SapA trimer + 33POPC)¹²⁺, and (SapA tetramer + 42POPC)¹⁶⁺ ions produced from modeling suggests that the solution structures are largely preserved in the gas phase, although the lipids do not maintain regular bilayer orientations