Mass spectrum of a pure peptide recorded in the positive ion mode is shown below:

(A) What is the reason for multiple peaks in the mass spectrum of a pure peptide?
(B) Which peak corresponds to the monoisotopic species of the peptide?
(C) What is the monoisotopic mass of the peptide?

Select the right answers from the options given below.

Correct option is C

The correct answer is Option (3):

(A) ¹³C isotope distribution, (B) Peak I, and (C) 1124.55 Da

Explanation:

(A) Reason for multiple peaks in mass spectrum of a pure peptide:

• In mass spectrometry, a pure peptide typically gives multiple peaks due to the presence of isotopic variants, primarily due to ¹³C isotope distribution.
• Most atoms in nature are a mixture of isotopes, and for carbon, the natural abundance is about 98.9% ¹²C and 1.1% ¹³C.
• As a peptide contains many carbon atoms, the chance of incorporation of one or more ¹³C isotopes increases, leading to M+1, M+2, M+3, etc. peaks, which are observed as successive higher m/z peaks.

(B) Monoisotopic species:

• The monoisotopic peak (Peak I) represents the peptide with only the most abundant (lightest) isotopes, i.e., all atoms as ¹²C, ¹H, ¹⁴N, ¹⁶O, etc.
• Hence, the first peak (1124.55 m/z) is the monoisotopic peak, and it is typically the leftmost and often highest or one of the highest intensity peaks.

(C) Monoisotopic mass:

• The m/z value of Peak I = 1124.55 Da corresponds to the monoisotopic mass of the peptide, as it is calculated solely from the lowest mass isotopes.

Information Booster:

1. Monoisotopic mass is the sum of the masses of the most abundant isotopes of all atoms in the molecule.
2. Isotopic distribution (like ¹³C, ¹⁵N, etc.) causes a predictable series of M+1, M+2… peaks in the mass spectrum.
3. The presence of ¹³C atoms increases the mass by 1 Da per ¹³C atom substituted.
4. The relative intensity of these peaks depends on the number of carbon atoms and their probabilities of being replaced by ¹³C.
5. In peptides with many carbon atoms, M+1 and M+2 peaks may sometimes be higher in abundance than M (monoisotopic).
6. Mass spectrometry in positive ion mode typically detects [M+H]⁺ ions.
7. The isotopic envelope helps in confirming molecular composition and verifying peptide purity.

Additional Information on Incorrect Options:

• Option 1 (Peak V as monoisotopic): Incorrect, Peak V is the M+4 peak, caused by multiple ¹³C substitutions.
• Option 2 and 4 (¹⁴C isotope distribution): ¹⁴C is a radioactive isotope, not naturally abundant, and does not cause this isotopic envelope in mass spectra.
• Hence, ¹³C is responsible, not ¹⁴C.