Expected Mass Table for Peptide Mass Spectrometry: Average vs Monoisotopic, Theoretical vs Observed

Monoisotopic mass is the mass of a molecule built only from the most abundant isotope of each element; average mass uses natural-abundance atomic weights and is the molecular weight printed on product pages. For BPC-157 (C62H98N16O22) the formula gives a monoisotopic mass of 1418.70 Da and an average mass of 1419.56 Da, both calculated; PubChem CID 9941957 records 1419.5 g/mol and the listing records 1419.53 g/mol. A mass spectrometry result on an analytical report compares an observed mass with one of these expected (theoretical) values, so whether the two "match" depends on which one is used and on how the instrument reports the ion. This page explains the terms, tabulates expected ions for the compounds listed in the catalog, computed from their recorded molecular formulas, and gives the masses each sequence shows after oxidation, water loss or deamidation.

What is the difference between monoisotopic and average mass?

Every element occurs as a mixture of isotopes. The monoisotopic mass is the mass of the molecule built entirely from the most abundant isotope of each element (¹²C, ¹H, ¹⁴N, ¹⁶O, ³²S). The average mass uses the natural-abundance-weighted atomic weights (C 12.011, H 1.008, N 14.007, O 15.999, S 32.06) and is the "molecular weight" printed on most product pages and reports. The gap grows with the number of carbon atoms, because the chance that a molecule carries one or more ¹³C atoms rises with size. Calculated from the recorded formulas, the gap is 0.21 Da for KPV (342 Da), 0.49 Da for DSIP (848 Da), 0.85 Da for BPC-157 (1,419 Da), 2.11 Da for sermorelin (3,358 Da) and about 3 Da for peptides of 4.9 to 5.4 kDa: 3.02 Da for TB-500, 3.14 Da for tesamorelin and 3.17 Da for FOXO4-DRI.

Which one an instrument reports depends on resolution. Low-resolution ESI or MALDI-TOF instruments see the isotope cluster as a single unresolved peak whose centroid corresponds to the average mass. High-resolution instruments resolve the individual isotope peaks; software then reports either the monoisotopic peak or a deconvoluted average, and the report should state which.

How close should the observed mass be to the theoretical mass?

The theoretical mass is calculated from the molecular formula; the observed mass is measured. Typical agreement is within ±0.1% for low-resolution ESI (about ±1.4 Da at 1,420 Da and ±5 Da at 5,000 Da) and within a few parts per million on high-resolution instruments. A discrepancy larger than the instrument tolerance means the ion measured is not the expected molecule: a deletion sequence (−57 Da for a missing glycine, −71 Da for alanine, and so on), an oxidized methionine (+16 Da), a deamidated asparagine or glutamine (+1 Da), a retained protecting group, or a different compound altogether. How these by-products arise during synthesis is covered in how research peptides are made.

How are charge states and m/z calculated?

Electrospray ionization produces multiply protonated ions, and a mass spectrometer measures the mass-to-charge ratio (m/z) rather than the mass. For an ion carrying z protons, m/z = (M + z × 1.00728) / z, where 1.00728 Da is the proton mass. Peptides with several basic residues commonly appear as [M+2H]²⁺ and [M+3H]³⁺ in ESI; the deconvoluted mass on the report has already been converted back to M. Sodium adducts (+21.98 Da relative to the protonated ion) and potassium adducts (+37.96 Da) also occur and are not sequence errors. Deconvolution, tolerances and the full reading order of an identity result are set out in mass spectrometry identity confirmation.

What masses should each listed compound show?

The table below is generated from the current catalog each time the page loads. Values are computed from the molecular formula of the free (neutral) peptide as recorded for each listed compound; the catalog molecular weight is shown alongside for comparison. Approximate m/z values use the computed average mass, or the catalog value where no formula is recorded. Counter-ions (TFA, acetate) are not part of the ion mass; see TFA vs acetate salts. Compounds without a parseable formula show a dash in the computed columns. CAS numbers and residue counts for the same compounds are in the CAS number and molecular weight table.

Computed columns use IUPAC standard atomic weights for the average mass and the principal-isotope masses for the monoisotopic mass. A difference between the computed average mass and the catalog molecular weight usually reflects rounding, a different set of atomic weights, or a catalog value that refers to a salt or complex rather than the free peptide.

Which modification masses can each listed compound show?

A modification mass is the calculated mass of the expected molecule after one specific chemical change, and it only applies where the sequence contains the residue that can change. Three common shifts are tabulated here: oxidation adds one oxygen atom (+15.99 Da) at methionine or tryptophan, and tryptophan can take up a second oxygen (+31.99 Da); water loss (−18.01 Da) follows aspartimide formation at an aspartic acid residue; deamidation (+0.98 Da) converts asparagine or glutamine to aspartic or glutamic acid, and the same +0.98 Da shift results when a C-terminal amide hydrolyses to the free acid. The table lists the calculated monoisotopic neutral mass for each shift, from the sequence and formula recorded for each of 17 listed peptides. A dash means the sequence has no residue for that shift. These are calculated reference values for reading a spectrum, not results for any production run.

All values are monoisotopic neutral masses in daltons, calculated from the formula in each compound's recorded identity data (C 12.000000, H 1.0078250, N 14.0030741, O 15.9949146, S 31.9720707). To read one as an ion, add 1.00728 Da for [M+H]⁺, or add 2 × 1.00728 Da and divide by 2 for [M+2H]²⁺. Where a sequence carries two or more of a residue, each extra modified residue adds the same shift again; MOTS-c, with two methionines and one tryptophan, can show +15.99, +31.99 and +47.98 Da. PT-141 and Melanotan-II carry norleucine in place of methionine; norleucine has no sulfur and takes no oxidation shift, so their only oxidation site is tryptophan. Selank and KPV contain none of these residues and are omitted, as are compounds without a confirmed sequence record.

How do you read the MS line on a report?

Look for three things: the expected value and whether it is stated as average or monoisotopic; the observed value and the instrument type (ESI, MALDI-TOF or high-resolution); and the difference, ideally expressed both in daltons and as a percentage or in ppm. Then check any extra peaks against the modification table above. Pepta Labs' analytical reports cover independent third-party HPLC purity analysis and mass-spectrometry identity confirmation, commissioned through the purchasing group on a sampled production run from the source; Pepta Labs does not test in-house. Because testing is commissioned by the purchasing group, the client field may name the group or source rather than Pepta Labs, and supplier-identifying fields are redacted. Vial labels identify the compound, quantity and research-use designation; they do not carry lot or batch numbers. Reports are sent by email on request, before you order if you want to see one first, and the reported HPLC purity is shown on each product page. For how purity and identity are measured, see HPLC vs mass spectrometry.

Frequently asked questions

How are the expected ions in this table computed?

Each ion is computed from the recorded molecular formula with m/z = (M + z × 1.00728) / z, where M is the neutral mass and 1.00728 Da is the proton mass. The generated table uses the average mass for its [M+H]⁺, [M+2H]²⁺ and [M+3H]³⁺ columns: for BPC-157 (C62H98N16O22, average 1419.56 Da calculated) that gives 1420.56, 710.79 and 474.19. On a high-resolution instrument the monoisotopic mass applies instead: 1418.70 Da, giving [M+H]⁺ 1419.71 and [M+2H]²⁺ 710.36.

What does a peak 16 Da heavier than the expected mass mean?

A peak 15.99 Da above the expected mass is the mass of one added oxygen atom, most often an oxidized methionine (methionine sulfoxide) or an oxidized tryptophan. It can only arise from a sequence that contains one of those residues. Among the compounds in the modification table, TB-500, MOTS-c, Semax, sermorelin and tesamorelin carry methionine, and DSIP, PT-141, Melanotan-I, Melanotan-II, MOTS-c and FOXO4-DRI carry tryptophan. BPC-157 has neither, so methionine or tryptophan oxidation cannot explain a +16 Da peak on a BPC-157 spectrum. Oxidation increases with air, light and moisture, which the peptide storage guide covers.

What does a peak 18 Da lighter than the expected mass mean?

A peak 18.01 Da below the expected mass is the loss of one water molecule. In a synthetic peptide a common source is aspartimide formation at an aspartic acid residue, a known side reaction of Fmoc solid-phase synthesis; an N-terminal glutamic acid cyclizing to pyroglutamate loses the same 18.01 Da, and some water loss can also occur inside the ion source during measurement. Of the compounds in the modification table, BPC-157 (two Asp), TB-500, DSIP, Livagen, sermorelin, tesamorelin and thymosin alpha-1 carry an aspartic acid with a free side chain; in PT-141 and Melanotan-II the aspartic acid side chain is bound in the ring lactam, so no aspartimide shift is listed for them.

Which ions should FOXO4-DRI show on an ESI spectrum?

FOXO4-DRI (C228H388N86O64, 5358.15 Da average) has 15 basic sites, its ten arginines, four lysines and the N-terminal amine, so ESI spreads it across a series of higher charge states rather than one peak. Calculated from the average mass, the 4+ ion falls at m/z 1340.54, 5+ at 1072.64, 6+ at 894.03, 7+ at 766.46, 8+ at 670.78, 9+ at 596.36 and 10+ at 536.82; the report's deconvoluted value should return to about 5358 Da (5354.98 Da monoisotopic). Its D-amino acids do not change the mass, since each D-residue has the same formula as its L-form.

Why can a DSIP report show masses near 864 and 880?

DSIP (WAGGDASGE, C35H48N10O15) has an N-terminal tryptophan, and tryptophan oxidation adds one oxygen (+15.99 Da) or two (+31.99 Da). From the monoisotopic mass of 848.33 Da, the calculated oxidized masses are 864.32 and 880.32 Da, seen as [M+H]⁺ at m/z 865.33 and 881.33; on an average-mass basis they sit near 864.8 and 880.8 Da. The Asp5 residue can also give an aspartimide peak 18.01 Da lighter, at 830.32 Da monoisotopic.

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