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Distinguishing Lysine and Glutamine in a Peptide


Lysine and glutamine are not easily distinguished by the most common approaches to peptide sequencing which involve mass spectrometers with low to moderate resolving power and low-energy collision-induced dissociation (CID). Lysine (C6H14N2O2 with a mass of 146.1055 u) and glutamine (C5H10N2O3 with a mass of 146.0691) differ by only 0.036 u. In this study, we demonstrate the measurement of a mixture of Substance P and a synthesized peptide (3-Gln ) with glutamine substituted for lysine in the Susbstance P sequence. Because the mass difference between Substance P and 3-Gln is 0.036 u, a resolving power of greater than 37,000 is required to separate each peptide. Additionally, we show that the TOF-TOF mode can be used to distinguish lysine and glutamine in these peptides by comparing the peak area ratio between a ions and d ions in the high-energy CID mass spectra.


Substance P standard was obtained from Sigma-Aldrich. The 3-Gln sample was synthesized and then provided by Hayashi Kasei.

  1. Substance P, Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Leu-Met (Sigma-Aldrich)
  2. 3-Gln, Arg-Pro-Gln-Pro-Gln-Gln-Phe-Phe-Leu-Met (Hayashi Kasei)

The peptide standard samples were dissolved in water containing 0.1% trifluoroacetic acid (TFA) at a concentration of 10 pmol/μL. α-Cyano-4-hydroxycinnamic acid (CHCA) was used as the matrix and was dissolved in 1:1 water/acetonitrile containing 0.1% TFA. Next, the peptide standard solution and CHCA solution were mixed together 1:1 by volume. Afterwards, 0.5 μL of this mixture was placed on the MALDI target plate (2.5 pmol/spot). Finally, the dried sample was measured using the SpiralTOF and TOF-TOF option available on the JMS-S3000 SpiralTOF MS system.

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