16 research outputs found
Use of top-down and bottom-up fourier transform ion cyclotron resonance mass spectrometry for mapping calmodulin sites modified by platinum anticancer drugs
Calmodulin (CaM) is a highly conserved, ubiquitous, calcium-binding protein; it binds to and regulates many different protein targets, thereby functioning as a calcium sensor and signal transducer. CaM contains 9 methionine (Met), 1 histidine (His), 17 aspartic acid (Asp), and 23 glutamine acid (Glu) residues, all of which can potentially react with platinum compounds; thus, one-third of the CaM sequence is a possible binding target of platinum anticancer drugs, which represents a major challenge for identification of specific platinum modification sites. Here, top-down electron capture dissociation (ECD) was used to elucidate the transition metal–platinum(II) modification sites. By using a combination of top-down and bottom-up mass spectrometric (MS) approaches, 10 specific binding sites for mononuclear complexes, cisplatin and [Pt(dien)Cl]Cl, and dinuclear complex [{cis-PtCl2(NH3)}2(μ-NH2(CH2)4NH2)] on CaM were identified. High resolution MS of cisplatin-modified CaM revealed that cisplatin mainly targets Met residues in solution at low molar ratios of cisplatin–CaM (2:1), by cross-linking Met residues. At a high molar ratio of cisplatin:CaM (8:1), up to 10 platinum(II) bind to Met, Asp, and Glu residues. [{cis-PtCl2(NH3)}2(μ-NH2(CH2)4NH2)] forms mononuclear adducts with CaM. The alkanediamine linker between the two platinum centers dissociates due to a trans-labilization effect. [Pt(dien)Cl]Cl forms {Pt(dien)}2+ adducts with CaM, and the preferential binding sites were identified as Met51, Met71, Met72, His107, Met109, Met124, Met144, Met145, Glu45 or Glu47, and Asp122 or Glu123. The binding of these complexes to CaM, particularly when binding involves loss of all four original ligands, is largely irreversible which could result in their failure to reach the target DNA or be responsible for unwanted side-effects during chemotherapy. Additionally, the cross-linking of cisplatin to CaM might lead to the loss of the biological function of CaM or CaM–Ca2+ due to limiting the flexibility of the CaM or CaM–Ca2+ complex to recognize target proteins or blocking the binding region of target proteins to CaM
Identification of Phosphatidylcholine Isomers in Imaging Mass Spectrometry Using Gas-Phase Charge Inversion Ion/Ion Reactions
Gas-phase
ion/ion reactions have been enabled on a commercial dual source, hybrid QhFT-ICR
mass spectrometer for use during imaging mass spectrometry experiments. These
reactions allow for the transformation of the ion type most readily generated
from the tissue surface to an ion type that gives improved chemical structural
information upon tandem mass spectrometry (MS/MS) without manipulating the
tissue sample. This process is demonstrated via the charge inversion reaction of
phosphatidylcholine (PC) lipid cations generated from rat brain tissue via
matrix-assisted laser desorption/ionization (MALDI) with 1,4-phenylenedipropionic
acid (PDPA) reagent dianions generated via electrospray ionization (ESI).
Collision induced dissociation (CID) of the resulting demethylated PC product anions
allows for the determination of the lipid fatty acyl tail identities and
positions, which is not possible via CID of the precursor lipid cations. The
abundance of lipid isomers revealed by this workflow is found to vary significantly
in different regions of the brain. As each isoform may have a unique cellular
function, these results underscore the importance of accurately separating and
identifying the many isobaric and isomeric lipids and metabolites that can
complicate image interpretation and spectral analysis.</p
Revealing Ligand Binding Sites and Quantifying Subunit Variants of Noncovalent Protein Complexes in a Single Native Top-Down FTICR MS Experiment
"Native" mass spectrometry (MS) has been proven to be increasingly useful for structural biology studies of macromolecular assemblies. Using horse liver alcohol dehydrogenase (hADH) and yeast alcohol dehydrogenase (yADH) as examples, we demonstrate that rich information can be obtained in a single native top-down MS experiment using Fourier transform ion cyclotron mass spectrometry (FTICR MS). Beyond measuring the molecular weights of the protein complexes, isotopic mass resolution was achieved for yeast ADH tetramer (147 kDa) with an average resolving power of 412,700 at m/z 5466 in absorption mode, and the mass reflects that each subunit binds to two zinc atoms. The N-terminal 89 amino acid residues were sequenced in a top-down electron capture dissociation (ECD) experiment, along with the identifications of the zinc binding site at Cys46 and a point mutation (V58T). With the combination of various activation/dissociation techniques, including ECD, in-source dissociation (ISD), collisionally activated dissociation (CAD), and infrared multiphoton dissociation (IRMPD), 40% of the yADH sequence was derived directly from the native tetramer complex. For hADH, native top-down ECD-MS shows that both E and S subunits are present in the hADH sample, with a relative ratio of 4:1. Native top-down ISD of the hADH dimer shows that each subunit (E and S chains) binds not only to two zinc atoms, but also the NAD/NADH ligand, with a higher NAD/NADH binding preference for the S chain relative to the E chain. In total, 32% sequence coverage was achieved for both E and S chains
Native Top-Down Electrospray Ionization-Mass Spectrometry of 158 kDa Protein Complex by High-Resolution Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
Fourier transform ion cyclotron resonance
mass spectrometry (FTICR
MS) delivers high resolving power, mass measurement accuracy, and
the capabilities for unambiguously sequencing by a top-down MS approach.
Here, we report isotopic resolution of a 158 kDa protein complex,
tetrameric aldolase with an average absolute deviation of 0.36 ppm
and an average resolving power of ∼520 000 at <i>m</i>/<i>z</i> 6033 for the 26+ charge state in magnitude
mode. Phase correction further improves the resolving power and average
absolute deviation by 1.3-fold. Furthermore, native top-down electron
capture dissociation (ECD) enables the sequencing of 168 C-terminal
amino acid (AA) residues out of 463 total AAs. Combining the data
from top-down MS of native and denatured aldolase complexes, a total
of 56% of the total backbone bonds were cleaved. The observation of
complementary product ion pairs confirms the correctness of the sequence
and also the accuracy of the mass fitting of the isotopic distribution
of the aldolase tetramer. Top-down MS of the native protein provides
complementary sequence information to top-down ECD and collisonally
activated dissociation (CAD) MS of the denatured protein. Moreover,
native top-down ECD of aldolase tetramer reveals that ECD fragmentation
is not limited only to the flexible regions of protein complexes and
that regions located on the surface topology are prone to ECD cleavage
Absorption-mode : the next generation of Fourier transform mass spectra
The Fourier transform spectrum can be presented in the absorption-mode (commonly used in FT-NMR), magnitude-mode (FT-ICR), and power-mode (engineering applications). As is routinely used in FT-NMR, it is well-known that the absorption-mode display gives a much narrower peak shape which greatly improves the spectrum; recently, the successful solution of the phase equation allowed broadband phase correction which makes it possible to apply the absorption-mode routinely in FT-ICR. With the empirical evidence provided herein, it has been confirmed that in addition to the improvement on resolving power, compared to the conventional magnitude-mode, the new absorption-mode improves the signal-to-noise ratio (S/N) of a spectrum by 1.4-fold and can improve the mass accuracy up to 2-fold with no extra cost in instrumentation. Therefore, it is worthwhile to apply and promote absorption-mode in routine FT-ICR experiments. © 2012 American Chemical Society
Variation of the Fourier Transform Mass Spectra Phase Function with Experimental Parameters
It has been known for almost 40 years that phase correction of Fourier transform ion cyclotron resonance (FTICR) data can generate an absorption-mode spectrum with much improved peak shape compared to the conventional magnitude-mode. However, research on phasing has been slow due to the complexity of the phase-wrapping problem. Recently, the method for phasing a broadband FTICR spectrum has been solved in the MS community which will surely resurrect this old topic. This paper provides a discussion on the data processing procedure of phase correction and features of the phase function based on both a mathematical treatment and experimental data. Finally, it is shown that the same phase function can be optimized by adding correction factors and can be applied from one experiment to another with different instrument parameters, regardless of the sample measured. Thus, in the vast majority of cases, the phase function needs to be calculated just once, whenever the instrument is calibrated
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
Absorption-Mode: The Next Generation of Fourier Transform Mass Spectra
The Fourier transform spectrum can be presented in the
absorption-mode
(commonly used in FT-NMR), magnitude-mode (FT-ICR), and power-mode
(engineering applications). As is routinely used in FT-NMR, it is
well-known that the absorption-mode display gives a much narrower
peak shape which greatly improves the spectrum; recently, the successful
solution of the phase equation allowed broadband phase correction
which makes it possible to apply the absorption-mode routinely in
FT-ICR. With the empirical evidence provided herein, it has been confirmed
that in addition to the improvement on resolving power, compared to
the conventional magnitude-mode, the new absorption-mode improves
the signal-to-noise ratio (S/N) of a spectrum by 1.4-fold and can
improve the mass accuracy up to 2-fold with no extra cost in instrumentation.
Therefore, it is worthwhile to apply and promote absorption-mode in
routine FT-ICR experiments