Tandem mass spectrometry for peptides: b and y ions explained

What do b, y and the other ion letters mean?

A single mass spectrum confirms that a peptide has the right total mass. Tandem mass spectrometry (MS/MS) goes further: it breaks the selected ion apart and measures the pieces. Because peptides tend to break at their amide bonds, the fragments form ladders whose mass differences spell out the sequence.

• Precursor ion: the intact, charged peptide selected for fragmentation.
• b ions: fragments that keep the N-terminus, numbered from the N-terminal end (b1, b2, b3…).
• y ions: fragments that keep the C-terminus, numbered from the C-terminal end (y1, y2, y3…).
• a, c, x and z ions: less common cleavage points on the same backbone, seen with other activation methods.
• The letters come from a 1984 proposal that is still the standard.

Source: Proposal for a common nomenclature for sequence ions in mass spectra of peptides, Biomed Mass Spectrom 1984 (PubMed 6525415)

How is a sequence read from the fragment ladder?

The mass difference between neighbouring ions in a series (b3 to b4, or y5 to y6) equals one amino acid residue: 57.02 Da for glycine, 71.04 for alanine, 128.09 for lysine, and so on. Reading consecutive differences gives the sequence in one direction from the b series and in the other from the y series; agreement between the two is what makes an assignment convincing.

Leucine and isoleucine have identical residue masses (113.08 Da), so standard collision-induced dissociation cannot tell them apart. Lysine (128.095) and glutamine (128.059) differ by only 0.036 Da and need high-resolution instruments.

Why do some peptides fragment unevenly?

Where a peptide breaks depends on where its protons sit. The 'mobile proton' model explains why peptides with arginine hold their charge tightly and fragment selectively, while peptides without basic residues fragment more evenly. Proline and acidic residues also produce characteristically strong cleavages.

Source: Mobile and localized protons: a framework for understanding peptide dissociation, J Mass Spectrom 2000 (PubMed 11180630); Utility of mass spectrometry for proteome analysis, part I, Expert Rev Proteomics 2008 (PubMed 19086863)

What does MS/MS add to a CoA?

• Distinguishes sequence isomers that a simple mass check cannot (for example two residues swapped).
• Locates a modification, such as an oxidised methionine, to a residue.
• Does not on its own measure purity; that remains a chromatographic figure.
• Cyclic and heavily modified peptides fragment less predictably and need expert interpretation.

How does Sable confirm identity?

Lots are released at ≥99% HPLC purity with identity confirmed by mass spectrometry. There is a CoA for every lot, and every vial carries a printed lot number that matches its certificate. A CoA is emailed within 2 hours of a request.

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