Reading a mass difference
A mass spectrometer measures mass to charge ratio very precisely. When the observed mass differs from the expected one, the size of the difference identifies what happened, because chemical modifications have exact and well-known masses.
The direction matters as much as the size. Oxidation adds 16, deamidation adds 1, a disulfide bridge removes 2, a C-terminal amide removes 1. A peptide 16 daltons lighter than expected has not been oxidised, whatever an unsigned comparison might suggest.
Parts per million and why it is the right unit
Mass accuracy is quoted in parts per million because instrument error scales with the mass being measured. An error of 0.01 daltons is excellent on a 1,000 dalton peptide and mediocre on a 100 dalton fragment.
As a rough guide, a high-resolution instrument such as an Orbitrap or a Q-TOF should give better than 5 ppm. A time-of-flight instrument typically manages 10 to 20. An ion trap or single quadrupole is working in units of daltons rather than parts per million.
Charge states and adducts
Electrospray ionisation produces multiply charged ions, so a 4,000 dalton peptide commonly appears at m/z values around 1,334, 1,001 and 801, corresponding to three, four and five protons. Each of those is the same molecule, and deconvolution is the process of recognising that.
Sodium and potassium adducts are the other routine confusion. A peak 22 daltons above the protonated species is sodium replacing a proton, not a modification of the peptide, and it is a sign of salt in the sample rather than a chemical change in the molecule.
- •Sodium adduct, plus 21.98 relative to the protonated form
- •Potassium adduct, plus 37.96
- •Ammonium adduct, plus 17.03
- •Residual trifluoroacetate, plus 113.99
Average against monoisotopic
A high-resolution instrument resolves the isotope pattern and reports the monoisotopic peak, the one where every atom is its lightest common isotope. A low-resolution instrument reports a centroid of the whole envelope, which is close to the average mass.
Comparing a monoisotopic measurement against an average calculated mass produces an apparent error that grows with molecular size: about 0.8 daltons at 1,400 and around three at 5,000. This is a units mismatch, not an instrument problem, and it is the first thing to check when a match is worse than the instrument should manage.
How the match is evaluated
A signed difference, an error in parts per million, and a lookup of the difference against a table of modification masses. Every step is signed, because the direction of a mass change is what identifies it.
difference (Da) = observed - expected error (ppm) = (observed - expected) / expected x 1,000,000 m/z = (neutral mass + z x 1.007276) / z match if |difference - modification delta| <= tolerance
- Take the signed difference. Observed minus expected. Keeping the sign is what distinguishes a mass gain from a mass loss, which is what identifies the modification.
- Express it in parts per million. The difference over the expected mass, scaled by a million. This is comparable across peptide sizes, which absolute daltons are not.
- Grade the match against instrument capability. Under 5 ppm is consistent with a high-resolution instrument, under 10 with most platforms, under 20 with a lower-resolution one. Beyond that, a different compound or a calibration problem is more likely than a good match.
- Search the modification table with the sign preserved. A modification is offered only when its own signed delta matches the observed signed difference within tolerance. The comparison used to be made on absolute values, so a mass loss of 16 was reported as possible oxidation.
- Compute m/z for the selected charge state. Neutral mass plus one proton per charge, divided by the charge. The proton mass used is 1.007276, the hydrogen atom less an electron.
What this method cannot tell you
- •It compares two numbers you supply. It cannot tell you whether the expected mass is right for the molecule you believe you have.
- •Several modifications share similar masses. Trimethylation and acetylation differ by 0.036 daltons, which only a high-resolution instrument separates.
- •It does not identify the position of a modification, only its mass. Locating it requires fragmentation.
- •A good mass match is consistent with identity but does not establish it. Isomers and rearrangements have identical masses.
Mass spec match calculator: frequently asked questions
A gain of 16 is the addition of one oxygen atom, almost always oxidation of a methionine to the sulfoxide, or occasionally of a tryptophan.
The direction is essential. A loss of 16 is not oxidation; it is something else entirely, and treating the two as equivalent is a genuine error rather than a shorthand.
Parts per million, the mass difference divided by the expected mass times a million. It is used because instrument error scales with the mass being measured.
- •Under 5 ppm: high-resolution instrument, high-confidence match
- •5 to 10 ppm: good on most platforms
- •10 to 20 ppm: acceptable on a lower-resolution instrument
- •Above 20 ppm: likely a different compound, an adduct, or a calibration problem
That is a sodium adduct: a sodium ion has replaced a proton, adding 21.98 daltons. The peptide itself is unchanged.
It indicates salt in the sample rather than a chemical modification. Desalting before analysis usually removes it.
Monoisotopic mass uses the lightest common isotope of every element; average mass uses the natural isotope-weighted average.
High-resolution instruments report monoisotopic; low-resolution ones report something close to average. The gap grows with size, from under a dalton at 1,400 to around three at 5,000.
Because electrospray ionisation produces multiply charged ions. Each charge state is the same molecule carrying a different number of protons.
A 4,000 dalton peptide typically shows up around m/z 1,334, 1,001 and 801 for three, four and five charges. Deconvolution software collapses the series back to one neutral mass.
Multiply the m/z by the charge, then subtract one proton mass, 1.007276, for each charge.
For m/z 1,001 at charge four: 1,001 times 4 is 4,004, less 4 times 1.007276, giving about 4,000 daltons.
Deamidation, where an asparagine or glutamine amide hydrolyses to a carboxylic acid. The exact shift is 0.984 daltons.
It is one of the most common degradation products in stored peptide solutions and is accelerated by higher pH and temperature.
A disulfide bridge has formed, removing two hydrogen atoms. The exact figure is 2.016 daltons per bridge.
Two bridges is 4.03 daltons. The mass tells you how many bridges formed but not which cysteines are paired.
It provides strong supporting evidence, not proof. A correct mass is consistent with the expected molecule and also with any isomer of it.
Confirming sequence requires fragmentation, where the peptide is broken into b and y ions and the pattern read against the expected series.
The window within which a known modification mass is considered to explain the observed difference. The default is 0.2 daltons.
Tighten it on a high-resolution instrument, where a 0.2 dalton window is far wider than the measurement error. Widen it on a single quadrupole, where it is not.
Because it compares signed values now. It used to compare absolute values, so a peptide 16 daltons lighter than expected was offered oxidation as an explanation for a change that adds mass.
Acetylation, at 42.011 daltons, usually at the N-terminus or on a lysine side chain.
Trimethylation is 42.047, a difference of 0.036 daltons that only a high-resolution instrument resolves. On a low-resolution one the two are indistinguishable.
Most often residual trifluoroacetate from reverse-phase purification, at 113.99 daltons, riding along as an adduct rather than as a covalent change.
It is a purification artefact and a sign that the salt exchange step was incomplete.
Not from the intact mass, which tells you only that something of a given mass was added or removed.
Locating it requires fragmentation: the b and y ion series shifts at the modified residue, and the position of the shift identifies it. The fragment mass calculator generates the expected series.
Because mass is not unique. Isomers have identical masses, and different compositions can coincidentally sum to nearly the same mass.
This is why identity confirmation combines mass with retention time and, where it matters, fragmentation.
Yes. Mass accuracy depends on calibration, and a systematic offset across all peaks in a run is the signature of a calibration that has drifted.
If everything on a spectrum is off in the same direction by a similar ppm figure, calibrate before interpreting any of it.
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