Quantitative protein sequencing using mass spectrometry: Use of low lonizing voltages in mass spectral analysis of methyl- and phenylthiohydantoin amino acid derivatives

doi:10.1016/0003-2697(72)90018-8

Analytical Biochemistry

Volume 45, Issue 1, January 1972, Pages 176-191

https://doi.org/10.1016/0003-2697(72)90018-8 Get rights and content

Abstract

The mass spectra of eighteen methylthiohydantoin and thirteen phenylthiohydantoin amino acid derivatives have been recorded at electron energies of 11, 20, and 70 electron volts. It is observed that the spectra of the majority of the derivatives studied were considerably reduced in complexity, containing in some cases only the molecular ion. In all the cases with three exceptions, the molecular ion was the base peak of the low-voltage spectrum. The loss of sensitivity at lower ionizing voltages was measured for a number of compounds and the sensitivity as measured by ion abundance was observed to be at a maximum around 20 eV and to decrease rapidly at lower energies. The use of low-energy electron impact ionization is compared to chemical ionization and the advantages and disadvantages discussed.

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Cited by (14)

Characterization of an electrospray ionization ion trap/linear time-of-flight mass spectrometer for phenylthiohydantoin-amino acid analysis
1998, International Journal of Mass Spectrometry
An electrospray ionization ion trap/time-of-flight mass spectrometer was evaluated as a detector for identification of phenylthiohydantoin (PTH)-amino acids, the final products in the Edman sequencing process of peptides and proteins. Each of 22 PTH-amino acids studied can be characterized by well-defined mass spectral patterns. Fragmentation from source excitation followed by a long trapping period (250 ms) gave rise to unique and intense fragmentation products. This allowed for the differentiation of isomers of PTH-leucine/PTH-isoleucine and PTH-α–aminobutyric acid/PTH-α–aminoisobutyric acid. The interpretation of the fragmentation patterns is presented. The detection limits and relative detection sensitivity of 20 standard PTH-amino acids were determined. It is shown that all these compounds can be detected at the 100 fmol level.
High sensitivity analysis of phenylthiohydantoin amino acid derivatives by electrospray mass spectrometry
1997, Journal of the American Society for Mass Spectrometry
A new methodology has been developed for high sensitivity electrospray ionization mass spectrometric analyses of phenylthiohydantoin (PTH) amino acid derivatives. Key components of the methodology are the use of a solvent system consisting of methanol/dichloromethane (1:1 v/v) containing 5-mM lithium triflate, a stainless steel electrode having a relatively large surface area, and a microscale electrospray nozzle that provides for stable electrospray at flow rates in the range of 100–500 nL/min. A linear response for the absolute signal intensity of the protonated molecule was observed for a number of derivatives over the concentration range of 50–1000 fmol/μL. For all except the arginine derivative, there was a decrease in the signal intensity with increasing flow rate with 100–300 nL/min being optimum. Collision induced dissociation (CID) product ion spectra were obtained for 21 derivatives including carboxymethyl cysteine and dehydrothreonine. Leucine and isoleucine can be distinquished on the basis of their CID product ion spectra. A subfemtomole detection limit was demonstrated for the phenylalanine PTH derivative in a selected reaction monitoring (SRM) experiment. Samples from an automated Edman microsequencer run have been analyzed using the new technique and compared to results obtained by conventional high-performance liquid chromatography analysis with UV detection. This work demonstrates the feasibility of using mass spectrometry to identify and quantitate the products generated by automated protein microsequencing using standard Edman degradation chemistry.
An electrospray ionization study of some novel alkylamine thiohydantoin amino acid derivatives
1995, Journal of the American Society for Mass Spectrometry
An electrospray ionization-mass spectrometric (ES-MS) study of some novel alkylamine thiohydantoin amino acid derivatives is presented. The alkylamine derivatives are being developed as part of an on-going effort to couple an Edmanlike protein sequencer to a bench-top electrospray ionization (ES) mass spectrometer. The ES-MS and capillary-skimmer collision-induced dissociation (CID) mass spectra of eight dimethylaminopropylthiohydantoin (DMAP-TH) amino acid derivatives as well as the trimethylaminopropylthiohydantoin (TMAP-TH), diethylaminopropylthiohydantoin, and dibutylaminopropylthiohydantoin (DBAP-TH) derivatives of Phe are presented. The spectra contain prominent [M + H]⁺ ions as well as fragment ions due to the loss of the respective neutral alkylamines. The CID spectrum of DMAP-TH-Phe also contains the dibutylaminoethyl cation. The relative responses of the alkylamine thiohydantoin derivatives obtained under identical solvent conditions are found to increase as the solvophobicity of the amino acid R group increases; the most solvophobic DBAP-TH-Phe gives rise to the highest overall response. DMAP-TH-Phe and the quaternary amine derivative TMAP-TH-Phe have comparable sensitivities when a sufficiently acidic solvent (pH = 3) is employed. Implications of these studies in the coupling of a protein sequencer to an ES mass spectrometer are discussed as are the requisite modifications of a single quadrupole mass spectrometer for ES analyses.
Analysis of phenylthiohydantoin amino acid mixtures for sequencing by thermospray liquid chromatography/mass spectrometry
1988, Analytical Biochemistry
Phenylthiohydantoin (PTH) amino acids, the derivatives of amino acids liberated in the course of automated N-terminal sequence analysis of peptides and proteins, are most commonly identified by high-performance liquid chromatography. This communication describes an extension to the methodology for PTH amino acid identification which exploits thermospray liquid chromatography/mass spectrometry for use in the confirmation of PTH amino acid identifications previously made solely on the basis of retention times. Thermospray mass spectra of the 19 synthetic PTH amino acids corresponding to the residues commonly observed during N-terminal sequencing have been acquired. These spectra show strong signals for the protonated molecular ion, accompanied in several cases by ions produced by limited fragmentation of the amino acid side chain and/or the PTH ring system. A reverse-phase separation protocol has been adapted for use with thermospray. The method permits recognition of the protonated molecular ions of all the standard PTH amino acids at the 150-pmol level on the basis of signalto-noise ratios of 10:1 or better with full scanning. The method has been tested on the N-terminal amino acid sequence analysis of 200 pmol of the standard protein β-lactoglobulin A, and has been found useful in the study of selected side-products of the sequencing chemistry.
A rapid and sensitive multisample identification method for methylthiohydantoin amino acids (MTH). Improved thin-layer chromatographic separation on a microscale
1976, Analytical Biochemistry
Two solvent systems have been established which are well suited for two-dimensional separation of 23 methylthiohydantoin-amino acids obtained in Edman degradation with methylisothiocyanate. Using a fluorescent indicator and doublefaced polyamide layers, it is also possible to separate and to identify at least 10 individual MTH¹ samples on a single 5 × 5 cm sheet at subnanomolar level (0.05–0.2 nmol) within 30 min by consecutively applying both solvents in identical direction. The methylthiohydantoins of chemically modified cysteine and methionine derivatives most commonly obtained in primary structure determination are included into the standard mixtures. In case of insufficient separation of the Leu, Ile, Phe, Val, and Thr derivatives, a third solvent was used. Synthesis and purification of MTH-AE¹-Cys, MTH-His, MTH-MetSO₂ and MTH-Ser are described. The new method is a valuable adjunct in manual and automated sequence determination.
Improved separation of the methyl- and phenylthiohydantoins of arginine, cysteic acid and histidine by micropolyamide thin-layer chromatography
1975, Journal of Chromatography A

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¹: NIH Research Career Development Awardee, and to whom correspondence should be addressed.

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Quantitative protein sequencing using mass spectrometry: Use of low lonizing voltages in mass spectral analysis of methyl- and phenylthiohydantoin amino acid derivatives

Abstract

Biochemistry

Z. Naturforsch

J. Amer. Chem. Soc

J. Amer. Chem. Soc