ionization-Fourier Transform Ion Cyclotron Resonance Research Articles

Analysis of enzymatically digested proteins and protein mixtures using a 9.4 Tesla Fourier transform ion cyclotron mass spectrometer

A commercially available 9.4 Tesla Fourier transform ion cyclotron resonance (FTICR) mass spectrometer was applied in the analysis of tryptic digests of protein mixtures without any separation. First, the method was demonstrated on a mixture of tryptic digests of equine cytochrome c, equine myoglobin and bovine serum albumin. The same method was then applied to human plasma from a healthy blood donor. Computer programs were employed to simplify analysis of the complex spectra. The 2745 peaks in the human plasma electrospray ionization FTICR spectrum could be reduced to 1165 isotopic clusters and 669 unique masses. Out of these, 82 masses matched tryptic fragments of serum albumin with mass measurement errors less than 10 ppm, covering 93% of the sequence. Another 16 masses were assigned to tryptic fragments of transferrin, covering 41% of the sequence on the 10 ppm mass measurement error level (14 within 2 ppm). The mass measurement errors were approximately normal distributed with a standard deviation of 1.7 ppm. This demonstrates the feasibility of combining the ultra-high mass resolving power and accuracy of FTICR mass spectrometry with automated computer analysis for investigating complex biological matrices.

Mass-selective ion accumulation and fragmentation in a linear octopole ion trap external to a fourier transform ion cyclotron resonance mass spectrometer

Electrosprayed protein ions are accumulated and mass selected in an external linear octopole trap before injection into a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. Mass selection is performed by application of superimposed rf and dc octopole electric potentials during ion accumulation. Ion trajectory stability and mass selection in the octopole field are explained qualitatively by analogy to a quadrupole mass filter. Accumulation of ions from a selected m/z range is demonstrated experimentally for 7 and 9.4 T electrospray ionization (ESI) FTICR mass spectrometers. Ion fragmentation in the octopole may occur under certain operating conditions.

Protein identification with a single accurate mass of a cysteine-containing peptide and constrained database searching.

A method for rapid and unambiguous identification of proteins by sequence database searching using the accurate mass of a single peptide and specific sequence constraints is described. Peptide masses were measured using electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry to an accuracy of 1 ppm. The presence of a cysteine residue within a peptide sequence was used as a database searching constraint to reduce the number of potential database hits. Cysteine-containing peptides were detected within a mixture of peptides by incorporating chlorine into a general alkylating reagent specific for cysteine residues. Secondary search constraints included the specificity of the protease used for protein digestion and the molecular mass of the protein estimated by gel electrophoresis. The natural isotopic distribution of chlorine encoded the cysteine-containing peptide with a distinctive isotopic pattern that allowed automatic screening of mass spectra. The method is demonstrated for a peptide standard and unknown proteins from a yeast lysate using all 6118 possible yeast open reading frames as a database. As judged by calculation of codon bias, low-abundance proteins were identified from the yeast lysate using this new method but not by traditional methods such as tandem mass spectrometry via data-dependent acquisition or mass mapping.

Characterization of the interface structure of enzyme-inhibitor complex by using hydrogen-deuterium exchange and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

We investigated the interaction between a thiol protease inhibitor, cystatin, and its target enzyme, papain, by hydrogen-deuterium (H/D) exchange in conjunction with successive analysis by collision-induced dissociation (CID) in an rf-only hexapole ion guide with electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS). The deuterium incorporation into backbone amide hydrogens of cystatin was analyzed at different time points in the presence or absence of papain, examining the mass of each fragment produced by hexapole-CID. In the absence of papain, amide hydrogens in short amino-terminal fragments, such as b10(2+) and b12(2+), were highly deuterated within 1 min. Although fewer fragments were observed for the cystatin-papain complex in the hexapole-CID spectra, significant reductions in initial deuterium content were recognized throughout the sequence of cystatin. This suggests that complex formation restricted the flexibility of the whole cystatin molecule. Detailed analyses revealed that a marked reduction in deuterium content in the region of residues 1-10 persisted for hours, suggesting that the flexible N-terminal region was tightly fixed in the binding pocket with hydrogen bonds. Our results are consistent with those of previous studies on the structure and inhibition mechanism of cystatin. We demonstrated here that enzyme-inhibitor interactions can be characterized by H/D exchange in combination with CID in a hexapole ion guide using ESI-FTICR MS rapidly and using only a small amount of sample.

Two-dimensional electrophoretic/chromatographic separations combined with electrospray ionization FTICR mass spectrometry for high throughput proteome analysis

A two-dimensional separation strategy combined with electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR) is being developed for high throughput proteomic analyses. Capillary isoelectric focusing (CIEF) coupled online with a robotic fraction collector is used to separate and collect microliter fractions of soluble Saccharomyces cerevisiae (yeast) proteins eluting from the capillary into a microtiter plate. Following tryptic digestion of each fraction, the resultant peptides are separated using capillary high performance liquid chromatography (HPLC) and analyzed by online ESI-FTICR. Protein identification is based upon the use of the experimentally measured peptide masses as accurate mass tags, augmented by conventional MS/MS methods as necessary, to identify proteins predicted from the yeast genome sequence. This new separation strategy is being evaluated using proteins extracted from yeast grown in natural isotopic abundance and 15N-enriched media. Two isotopically distinct versions of each peptide are thus observed in the ESI-FTICR spectra. The mass differences between the two versions are used to determine the number of nitrogen atoms in the peptide, and provide an additional constraint that aids protein identification. More importantly, the use of this stable-isotope labeling strategy enables the generation of “comparative displays” of the precise relative protein abundances. This two-dimensional separation strategy combined with ESI-FTICR analysis is expected to be highly amenable to automation. © 2000 John Wiley & Sons, Inc. J Micro Sep 12: 383–390, 2000

Analysis of enzymatically digested proteins and protein mixtures using a 9.4 Tesla Fourier transform ion cyclotron mass spectrometer

A commercially available 9.4 Tesla Fourier transform ion cyclotron resonance (FTICR) mass spectrometer was applied in the analysis of tryptic digests of protein mixtures without any separation. First, the method was demonstrated on a mixture of tryptic digests of equine cytochrome c, equine myoglobin and bovine serum albumin. The same method was then applied to human plasma from a healthy blood donor. Computer programs were employed to simplify analysis of the complex spectra. The 2745 peaks in the human plasma electrospray ionization FTICR spectrum could be reduced to 1165 isotopic clusters and 669 unique masses. Out of these, 82 masses matched tryptic fragments of serum albumin with mass measurement errors less than 10 ppm, covering 93% of the sequence. Another 16 masses were assigned to tryptic fragments of transferrin, covering 41% of the sequence on the 10 ppm mass measurement error level (14 within 2 ppm). The mass measurement errors were approximately normal distributed with a standard deviation of 1.7 ppm. This demonstrates the feasibility of combining the ultra-high mass resolving power and accuracy of FTICR mass spectrometry with automated computer analysis for investigating complex biological matrices.

Observation of hydrogen-deuterium exchange of ubiquitin by direct analysis of electrospray capillary-skimmer dissociation with Fourier transform ion cyclotron resonance mass spectrometry.

The structure of ubiquitin, a small cytoplasmic protein with an extended beta-sheet and an alpha-helix surrounding a hydrophobic core, has been characterized by hydrogen-deuterium (H/D) exchange labeling in conjunction with successive analysis by capillary-skimmer dissociation with electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS). The deuterium content of each fragment ion was investigated at different times, and the results indicate that the deuterium incorporation rate into the backbone amides of ubiquitin varied depending on the environment of the amide hydrogens. Amide hydrogens of the N-terminal beta-strand showed quite slow exchange while those of the 35-39 loop were exchanged within a short exposure time to deuterium oxide. It was also possible to evaluate the difference in hydrogen-bond stability. The present data are consistent with the structural features obtained by X-Ray and NMR analyses. Although some of the labeling information might be lost by the scrambling of amide protons during capillary-skimmer dissociation, the results demonstrate that the present method provides useful higher-order structural information for proteins.

Probing RegA/RNA Interactions Using Electrospray Ionization–Fourier Transform Ion Cyclotron Resonance–Mass Spectrometry

The interactions of bacteriophage T4 regA protein, a unique translational regulator, with RNAs of various size and sequence were studied using electrospray ionization–Fourier transform ion cyclotron resonance–mass spectrometry. Using very gentle interface conditions, regA/RNA complexes with a 1:1 binding stoichiometry were observed for all four target RNAs studied, consistent with solution binding studies. Competitive binding of target RNAs and their degradation products with regA demonstrated that the loss of a single nucleotide resulted in a dramatic change in binding affinity in some cases. Competitive binding of regA with four target RNAs revealed similar relative binding affinity order to that suggested by previousin vitrorepression experiments. The use of sustained off-resonance irradiation for collisionally induced dissociation of a regA/RNA complex suggested the potential for directly obtaining information regarding the regA binding domain.

Distinguishing Small Molecular Mass Differences of Proteins by Mass Spectrometry

Electrospray ionization–Fourier transform ion cyclotron resonance (ESI–FTICR) mass spectrometryallows for high-resolution, accurate mass analysisof multiply charged ions of proteins. In the workdescribed here, the ability of ESI–FTICR to distinguish small differences in molecular mass is evaluated. Ubiquitin was used as an internal mass calibration standard to measure the molecular mass of cytochromec, myoglobin, and several carbonic anhydrase isoforms. Mass calibration was based onthe tallest isotopic peak of each ubiquitin chargestate. Ubiquitin performed well as an internal standard because its charge states covered the appropriate mass range, interference was minimal, and thetallest peak was easily identified. The peak massesof cytochrome c (12.5 kDa) and myoglobin (17 kDa) were measured to an accuracy of about 0.02 Da (<2ppm). However, errors of 1.0 Da were observedfor some individual determinations because of the difficulty in identifying the tallest peak. When the technique was applied to bovine carbonic anhydrase II, even combining data from several charge statesdid not yield an unequivocal assignment of thetallest peak, resulting in a mass assignment of 29,023.7 or 29,024.7. Similarly, measurements of two isoforms with a mass difference of 1 Da, human carbonic anhydrase I, pI6.0 and 6.6, yielded overlapping values for the mass of the tallest peak. However, these two isoforms were clearly distinguished by (a) identification of the tallest peak using a measurement of average mass as a guide and (b) comparison of the isotopic peak intensity patterns.

Assessing enzyme substrate specificity using combinatorial libraries and electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry

A model experiment for the ‘on-line’ screening of substrate libraries by enzymes using combinatorial libraries in combination with electrospray ionization-Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry has been performed. The reaction between the electrophilic substrate 1-chloro-2,4-dinitrobenzene and components of a H-γ-Glu-Cys-Xxx-OH library, catalyzed by glutathione-S-transferase, has been monitored. It shows the feasibility of ‘two-dimensional’ screening of substrate libraries by ESI-FTICR mass spectrometry. © 1997 John Wiley & Sons, Ltd.

Assessing enzyme substrate specificity using combinatorial libraries and electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry.

A model experiment for the 'on-line' screening of substrate libraries by enzymes using combinatorial libraries in combination with electrospray ionization-Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry has been performed. The reaction between the electrophilic substrate 1-chloro-2,4-dinitrobenzene and component of a H-gamma-Glu-Cys-Xxx-OH library, catalyzed by glutathione-S-transferase, has been monitored. It shows the feasibility of 'two-dimensional' screening of substrate libraries by ESI-FTICR mass spectrometry.

Fourier transform ion cyclotron resonance studies of lanthanide(III) porphyrin-phthalocyanine heteroleptic sandwich complexes by using electrospray ionization

A number of neutral Ianthanide(III) porphyrin-phthalocyanine heteroleptic sandwich complexes have been studied by using positive-ion electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry. The investigated compounds are represented as LnIII(TPP)(Pc), Ln 2 III (TPP)(Pc)2, and Ln 2 III (TPP)2(Pc), where Ln = Sm, Eu, or Gd, TPP is 5,10,15,20-tetraphenylporphyrinate, and Pc is phthalocyaninate. In all cases, intense signals corresponding to the singly charged molecular radical cations are observed. The formation of these molecular radical cations in electrospray ionization is attributed to electrochemical oxidation at the electrospray needle. Multiply charged molecular ions up to +5 are also observed. They are tentatively assigned to be formed from successive oxidation of the ligand(s). Apart from the molecular weight information, tandem mass spectrometry offers additional structural information on these complexes. From the fragmentation pattern of the europium complexes under collision-induced dissociation conditions, the configurations of the triple-decker complexes are assigned mass spectrometrically to be (TPP)Eu(Pc)Eu(Pc) and (TPP)Eu(Pc)Eu(TPP). In comparison with the previous spectroscopic findings that the positive “hole” is localized in the ligands of the complexes, there is evidence to suggest that intramolecular charge transfer or hole delocalization does occur within the macrocycles or between the metal centers and the macrocycles before fragmentation. The occurrence of this charge-transfer process is tentatively attributed to the result of collisional activation.

Characterization of PCR products from bacilli using electrospray ionization FTICR mass spectrometry.

A procedure for rapid purification of polymerase chain reaction (PCR) products allowing precise molecular weight determination using electrospray ionization-Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry is described. PCR amplification utilized the DNA polymerase from Pyrococcus furiosus (Pfu) which, unlike Taq, does not incorporate a nontemplated terminal deoxyadenosine phosphate. An 89-base pair nucleotide portion of the spacer region between the 16S and 23S ribosomal rRNA genes was amplified from the genome of three members of Bacillus cereus group and a 114 nucleotide region from the Bacillus subtilis. PCR involves polymerization of nucleotide precursors using two oligonucleotide primers and an amplification enzyme, as well as the presence of metal ions. Mass spectrometric analysis greatly benefits from removal of the oligonucleotide primers (15- and 17-mers in this instance) and nucleotide precursors since they adversely affect sensitivity and metal ion adduction results in an inaccurate molecular weight determination. In the presence of guanidinium hydrochloride the PCR products bind preferentially to a silica resin, allowing removal of other components (i.e., dNTP's primers, and salts). Further removal of metal ions was accomplished using a microdialysis device, allowing samples to be pumped through a hollow cellulose fiber with external countercurrent flow of 2.5 mM ammonium acetate. Prior to injection into the mass spectrometer, the sample buffer was adjusted to 50 vol % acetronitrile, 25 mM piperidine, and 25 mM imidazole, which enhanced signal intensity. The molecular weights of the PCR products determined by nucleotide sequence and MS analysis were in excellent agreement, and several PCR products were analyzed where mass differences corresponding to single base substitutions could be accurately assigned. These assignments were possible due to the high mass precision, accuracy, and resolution FTICR inherently affords. This constitutes the first report demonstrating the ionization and detection of PCR products by mass spectrometry with mass precision and accuracy for assignment of such modifications or substitutions.

Positive-ion Electrospray-Fourier Transform Ion Cyclotron Resonance Mass Spectra of Polypeptides and Proteins

We have determined the mass spectra of three polypeptides which have been designed as an effective reagent to be used in the diagnosis of a Chinese strain of Hepatitis E Virus (HEV). The spectra were determined by electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometer. These are peptide 30, peptide 33 and peptide 42, where the guanidine amino group of arginine in each polypeptide was protected. The molecular weights were respectively measured as 3575, 4094 and 5390 Dalton, against the molecular weights of bovine serum albumin (66430 Da) and bovine insulin (5732.77 Da).

Analysis of Double-stranded Polymerase Chain Reaction Products from the Bacillus cereusGroup by Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

The analysis of polymerase chain reaction (PCR) products by electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR) has been achieved. Specifically, a 105 base-pair nucleotide portion of the ribosomal spacer region was amplified in two members of the B. cereus group (i.e. B. thuringiensis and B. cereus) using PCR. These amplified regions were then analyzed by gel electrophoresis and ESI-FTICR. Based on the predicted sequence of the PCR products for each organism, the mass measurement using ESI-FTICR matched the theoretical mass within experimental error and was consistent with gel electrophoresis results. In contrast, for the typical several hour time-scale of the gel electrophoresis experiment, the mass spectrometric analysis was completed in a matter of minutes. To our knowledge, this constitutes the first report demonstrating the ionization and detection of a double-stranded PCR product by ESI-MS. This preliminary result indicates the potential use of ESI-MS to analyze PCR products on a rapid time-scale, with potential for medical and taxonomic applications.

Analysis of double-stranded polymerase chain reaction products from the Bacillus cereus group by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

The analysis of polymerase chain reaction (PCR) products by electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR) has been achieved. Specifically, a 105 base-pair nucleotide portion of the ribosomal spacer region was amplified in two members of the B. cereus group (i.e. B. thuringiensis and B. cereus) using PCR. These amplified regions were then analyzed by gel electrophoresis and ESI-FTICR. Based on the predicted sequence of the PCR products for each organism, the mass measurement using ESI-FTICR matched the theoretical mass within experimental error and was consistent with gel electrophoresis results. In contrast, for the typical several hour time-scale of the gel electrophoresis experiment, the mass spectrometric analysis was completed in a matter of minutes. To our knowledge, this constitutes the first report demonstrating the ionization and detection of a double-stranded PCR product by ESI-MS. This preliminary result indicates the potential use of ESI-MS to analyze PCR products on a rapid time-scale, with potential for medical and taxonomic applications.

Selected ion accumulation of noncovalent complexes in a fourier transform ion cyclotron resonance mass spectrometer

AbstractAn electrospray ionization–Fourier transform ion cyclotron resonance (ESI–FTICR) mass spectrometer has been used in conjunction with the technique of selected‐ion accumulation (SIA) to investigate its utility for the study of the gas phase behavior of noncovalent complexes from solution. SIA is demonstrated to provide the selective accumulation of weakly bound species, providing a significant enhancement in the attainable signal‐to‐noise ratio and dynamic range. The frequency response of quadrupole excitation was first determined by selectively accumulating single charge states of large, highly adducted, highly charged molecules such as bovine albumin and bovine albumin dimer. Under the conditions employed, the response (selective accumulation) was found to be as narrow as 500 Hz. Additionally the SIA was found to be sufficiently gentle to allow accumulation of both specific and nonspecific weak noncovalent complexes, such as the myoglobin–heme complex observed in basic solutions and complexes of myoglobin and single amino acids, such as tryptophan. This result is probably due to the continual application of quadrupole excitation during the high pressure accumulation event, preventing any significant growth of magnetron motion, and subsequently, allowing interconversion to form only small cyclotron radii. We also demonstrate the simultaneous application of SIA with dipolar sustained off‐resonance irradiation (SORI) during the ion accumulation period so as to allow selective accumulation of dissociation products. The SIA/SORI combination promises to greatly extend (MS)n capabilities by allowing the selective accumulation of any dissociation product in any step along a given dissociation pathway.