High-resolution accurate mass measurements of biomolecules using a new electrospray ionization ion cyclotron resonance mass spectrometer

Winger, Brian E.; Hofstadler, Steven A.; Bruce, James E.; Udseth, Harold R.; Smith, Richard D.

doi:10.1016/1044-0305(93)85018-s

Cited by 168 publications

(116 citation statements)

References 39 publications

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“…All experiments were performed by using a previously described external ion source 7 tesla FTICR mass spectrometer (32). Briefly, the instrument is equipped with three serial quadrupoles, which are separated by two conductance limits and are operated synchronously, to guide ions produced by the ESI source to a rectangular closed cell through four stages of differential pumping.…”

Section: Msmentioning

confidence: 99%

Direct mass spectrometric analysis of intact proteins of the yeast large ribosomal subunit using capillary LC/FTICR

Lee

Berger

Martinović

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry coupled with capillary reverse-phase liquid chromatography was used to characterize intact proteins from the large subunit of the yeast ribosome. High mass measurement accuracy, achieved by ''mass locking'' with an internal standard from a dual electrospray ionization source, allowed identification of ribosomal proteins. Analyses of the intact proteins revealed information on cotranslational and posttranslational modifications of the ribosomal proteins that included loss of the initiating methionine, acetylation, methylation, and proteolytic maturation. High-resolution separations permitted differentiation of protein isoforms having high structural similarity as well as proteins from their modified forms, facilitating unequivocal assignments. The study identified 42 of the 43 core large ribosomal subunit proteins and 58 (of 64 possible) core large subunit protein isoforms having unique masses in a single analysis. These results demonstrate the basis for the high-throughput analyses of complex mixtures of intact proteins, which we believe will be an important complement to other approaches for defining protein modifications and their changes resulting from physiological processes or environmental perturbations.M ass spectrometry has evolved into a powerful tool for analyzing biomolecules because of the development of matrix-assisted laser desorption ionization (1, 2) and electrospray ionization (ESI) (3) and advances in both mass analyzers and data processing capabilities. With these ionization methods, which are amenable to megadalton-size molecules, the broadly useful detection and characterization of biopolymers by mass spectrometric methods are feasible. For example, MS has become a preferred analytical tool for proteome analyses, in which dynamic populations of proteins are identified and quantified. Proteins are now routinely identified by mass spectrometric and tandem mass spectrometric analysis of proteolytic digests of individual protein spots on two-dimensional (2D) PAGE (4). However, the intact protein level analyses of complex protein mixtures, while potentially providing complementary and direct information for protein identification, have been a much greater experimental challenge and only rarely attempted (5, 6).Many pivotal cellular processes are not carried out by individual proteins, but rather by large protein-protein complexes and protein-nucleic acid complexes. The analyses of such complexes have been greatly expanded by improvements in both biological separations and MS. One of the more complicated protein complexes yet studied by MS is the cytosolic ribosome complex. Efforts to define the protein composition (7, 8), modifications (9), and subunit interactions (10) of ribosomes generally have mirrored the development of biomolecular analysis techniques and experimental approaches for the study of noncovalent protein complexes.Ribosomes are the canonical example of ribonucleoprotein complexes in both prokaryot...

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Section: Msmentioning

confidence: 99%

Direct mass spectrometric analysis of intact proteins of the yeast large ribosomal subunit using capillary LC/FTICR

Lee

Berger

Martinović

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

183

133

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“…The other instrument used was a 7-T Fourier transform ion cyclotron resonance mass spectrometer (FTICR) (IonSpec, Irvine, CA) with a modified Analytica (Branford, CT) ESI source (36 2 for selected species.…”

mentioning

confidence: 99%

Direct measurement of oligonucleotide binding stoichiometry of gene V protein by mass spectrometry.

Cheng

Harms²,

Goudreau³

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

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The binding stoichiometry of gene V protein from bacteriophage ft to several oligonucleotides was studied using electrospray ionization-mass spectrometry (ESI-MS). Using mild mass spectrometer interface conditions that preserve noncovalent associations in solution, gene V protein was observed as dimer ions from a 10 mM NH4OAc solution.Addition of oligonucleotides resulted in formation of proteinoligonucleotide complexes with stoichiometry of approximately four nucleotides (nt) per protein monomer. A 16-mer oligonucleotide gave predominantly a 4:1 (protein monomer: oligonucleotide) complex while oligonucleotides shorter than 15 nt showed stoichiometries of 2:1. Stoichiometries and relative binding constants for a mixture of oligonucleotides were readily measured using mass spectrometry. The binding stoichiometry of the protein with the 16-mer oligonucleotide was measured independently using size-exclusion chromatography and the results were consistent with the mass spectrometric data. These results demonstrate, for the first time, the observation and stoichiometric measurement of proteinoligonucleotide complexes using ESI-MS. The sensitivity and high resolution of ESI-MS should make it a useful tool in the study of protein-DNA interactions.Protein-DNA interactions are involved in many cellular processes and thus are fundamentally important for the functioning of biological systems. The stoichiometry of binding is an important characteristic of protein-DNA interactions. Determination of binding stoichiometries can be difficult when there are mixed stoichiometries present in a sample or when molecular mass (Mr) differences between possible alternative stoichiometries are small (1). Mass spectrometry (MS) is the most accurate method for Mr measurement, and thus is potentially useful for accurate determination of protein-DNA binding stoichiometry. However, the application of MS to the direct characterization of protein-DNA complexes has been precluded due to the difficulty of transferring these complexes intact from solution into the mass spectrometer using conventional ionization methods. Electrospray ionization (ESI) allows ionization of non-volatile samples from solution and the development of this technique has greatly expanded the application of mass spectrometry in the analysis of biomolecules (2-5). Recently, a number of groups have reported observations of specific noncovalent complexes of biomolecules from solution using ESI-MS (6-11). These reports prompted us to investigate the application of this technique to intact protein-DNA noncovalent complexes.We chose gene V protein from bacteriophage fl for our study due to its well characterized structure (12-16) and the extensive studies on the DNA binding stoichiometry and cooperatively (17)(18)(19)(20)(21)(22). The gene V protein stabilizes singlestranded DNA (ssDNA) during phage replication, and shifts the synthesis of phage DNA from double-stranded form to single-stranded form, in the. later stage of phage infection, by preventing the synthesis of th...

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“…Whereas the monoisotopic peak of mass spectrometry may be below the noise level and higher isotopes may dominate completely. For most biological macromolecules with molecular weight greater than 10 kDa, the monoisotopic relative abundance is so low that almost undetectable [6], because the tiny peak is buried in the background noise in the spectrum. For isotopic depleted biomolecules, the monoisotopic peak is much higher in abundance and easily recognized by its sudden onset.…”

Section: Resultsmentioning

confidence: 99%

<b>THE MASS OF CELLULAR RETINOIC ACID BINDING PROTEIN I INVESTIGATED BY <sup>13</sup>C DEPLETION AND MASS SPECTROMETRY</b>

Zhang

Song

Wen

2009

Bull. Chem. Soc. Eth.

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ABSTRACT. The accuracy of mass spectrometry used to determine large molecular mass as proteins is often influenced by the isotopic compositions within a protein. Isotopic depletion is a powerful tool to resolve this problem. Using Fourier transform ion cyclotron resonance mass spectrometer, we investigated the 13 C depleted cellular retinoic acid binding protein I, the measured mass accuracy is increased compared with the natural protein.

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High-resolution accurate mass measurements of biomolecules using a new electrospray ionization ion cyclotron resonance mass spectrometer

Cited by 168 publications

References 39 publications

Direct mass spectrometric analysis of intact proteins of the yeast large ribosomal subunit using capillary LC/FTICR

Direct mass spectrometric analysis of intact proteins of the yeast large ribosomal subunit using capillary LC/FTICR

Direct measurement of oligonucleotide binding stoichiometry of gene V protein by mass spectrometry.

<b>THE MASS OF CELLULAR RETINOIC ACID BINDING PROTEIN I INVESTIGATED BY <sup>13</sup>C DEPLETION AND MASS SPECTROMETRY</b>

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