Molecular mass of a biomolecule is characterized in mass spectroscopy by the monoisitopic mass M~mono~ and the average isotopic mass M~av~. We found that peptide masses mapped on a plane made by two parameters derived from M~mono~ and M~av~ form a peculiar global feature in form of a band-gap 5-7 ppm wide stretching across the whole peptide galaxy, with a narrow (FWHM 0.2 ppm) line in the centre. The a priori probability of such a feature to emerge by chance is less than 1:100. Peptides contributing to the central line have elemental compositions following the rules S=0; Z = (2C - N - H)/2 =0, which nine out of 20 amino acid residues satisfy. The relative abundances of amino acids in the peptides contributing to the central line correlate with the consensus order of emergence of these amino acids, with ancient amino acids being overrepresented in on-line peptides. Thus the central line is a relic of ancient life, and likely a signature of its emergence in abiotic synthesis. The linear correlation between M~av~ and M~mono~ reduces the complexity of polypeptide molecules, which may have increased the rate of their abiotic production. This, in turn may have influenced the selection of these amino acid residues for terrestrial life. Assuming the line feature is not spurious, life has emerged from elements with isotopic abundances very close to terrestrial levels, which rules out most of the Galaxy

Alexander  R. Zubarev

Corina Mayrhofer

Eva Y. M. Fung

Konstantin Artemenko

Roman A. Zubarev

English

Nature Precedings

1 
Early Life Relict Feature in Peptide Mass Distribution  
Roman A. Zubarev*, Konstantin A. Artemenko, Alexander R. Zubarev,  
Corina Mayrhofer & Y. M. Eva Fung 
Molecular Biometry, Department for Cell and Molecular Biology, Uppsala University, 
Box 596,  SE-75 124 Uppsala, Sweden 
 ABSTRACT 
Molecular mass of a biomolecule is characterized in mass spectroscopy by the 
monoisitopic mass Mmono and the average isotopic mass Mav. We found that peptide 
masses mapped on a plane made by two parameters derived from Mmono and Mav form a 
peculiar global feature in form of a ‘band gap’ 5-7 ppm wide stretching across the 
whole ‘peptide galaxy’, with a narrow (FWHM ≈0.2 ppm) ‘line’ in the centre. The a 
priori probability of such a feature to emerge by chance is less than 1:100. Peptides 
contributing to the central line have elemental compositions following the rules S=0; Z 
= (2C - N – H)/2 =0, which nine out of 20 amino acid residues satisfy.  The relative 
abundances of amino acids in the peptides contributing to the central line correlate with 
the consensus order of emergence of these amino acids, with ancient amino acids being 
overrepresented in on-line peptides.  Thus the central line is a relic of ancient life, and 
likely a signature of its emergence in abiotic synthesis. The linear correlation between 
Mav and Mmono reduces the complexity of polypeptide molecules, which may have 
increased the rate of their abiotic production. This, in turn may have influenced the 
selection of these amino acid residues for terrestrial life.  Assuming the line feature is 
not spurious, life has emerged from elements with isotopic abundances very close to 
terrestrial levels, which rules out most of the Galaxy. 
2 
 Molecular mass (MM) of a biopolymer is a distribution containing information 
on the exact masses and abundances of all stable isotopes of the constituent elements. 
For polypeptides, the five constituent elements C, H, O, N and S encompass 12 stable 
isotopes, the five lightest isotopes being dominant. The full description of the peptide 
MM is thus a combination of the five variable coefficients of a brutto-formula (c, h, o, n 
and s), 12 constant isotope masses and seven relative abundances of less abundant 
isotopes, which are also variable. In biomolecular mass spectroscopy, the traditional 
approach is the assumption of constant isotopic compositions.  MM of a biopolymer is 
then considered to have two constant values, the monoisotopic mass Mmono and the 
average isotopic mass Mav.  Reduction of dimensionality to two affords easy 
visualization on a two dimensional (2D) plot using derivatives of the two mass values as 
axes. One convenient mass derivative is the monoisotopic mass defect,  
ΔMm = Mmono – Mnom,        (1a) 
where Mnom is the nominal (integer) mass (e.g. 14N = 14, 32S = 32, etc.). ΔMm is related 
to the binding energy of the nucleons in the atomic nucleus, and for each element it is a 
strict constant. The other derivative is the isotopic mass shift  
ΔMis = Mav – Mnom        (1b) 
determined by the relative abundances of the less abundant isotopes.  Since heavier 
isotopes can be enriched or depleted in physico-chemical process, ΔMis is a constant 
only approximately. ΔMis values are tabulated, and for biogenic elements C, H, N and O 
are constant within 1-3% (with a bigger variation for H) for most terrestrial organic 
molecules. To eliminate the mass dependence, introduce  
NMD = 1000•ΔMm/Mnom      (2a) 
NIS= 1000•ΔMis/Mnom       (2b) 
3 
where NMD [‰] is the normalized monoisotopic defect and NIS [‰] is the normalized 
isotopic shift, respectively. NMD and NIS can be considered independent mass 
parameters for peptides with arbitrary amino acid compositions. 
Mapping theoretical NMD and NIS values of 3600 tryptic peptides revealed an 
unexpected global feature in form of a ‘band gap’ across the ‘peptide galaxy’, with a 
narrow line in the centre (Figure 1). The best fit to the Line of a linear equation: 
 NIS = a*RMD + b        (3) 
gave a = 0.350574 and b = 0.395622 ‰ with R2=0.99999.  The distance d from the dots 
to the Line is shown in Figure 2, with the Line now represented by a peak at d = 0 ppm. 
No dots outside the central peak are found in the region between -3.0 ppm and +2.5 
ppm. The molecules on the Line have elemental compositions obeying the rule: 
 s = 0;   h = 2c – n.       (4)  
For sulfur-free peptides (i.e. no Met or Cys), the factor  
z = (2c - n – h)/2       (5) 
reflects the deviation of the elemental composition from the rule (4).  Unlike both NMD 
and NIS, z is an additive parameter, z[A+B] = z[A] + z[B]. The position on the 2D mass 
map depends upon the z-value: molecules with z<0 lie under the Line, while those with 
z>0 occupy the region above the Line.   
While masses of peptides with z=0 are mapped onto the Line, those with z=±1 
contribute to broader distributions on both sides of it (Figure 3), with the peptides with 
z=±2 forming even broader distributions farther away. The discrete nature of z is the 
reason for the gap between the Line and neighbouring points with z=±1.  The peptide 
masses with z=n can be focussed into the Line by adding/subtracting nH2Ox to the 
elemental formula, as shown in Figure 3. The number of oxygens x can be arbitrary, and 
4 
will only affect the position on the Line. Note that the addition/subtraction of nH2Ox 
does not alter the Line position, and only moves the peptide masses on the 2D plot.  
According to Table 1, nine out of 18 sulfur-free residues have z =0, i.e. obey the 
rule (4). Acidic residues Glu and Asp have a z = 1. Since only basic Lys and Arg have 
negative z-values, peptides with z<0 must contain more than one basic residue. Thus, if 
these peptides are produced by trypsinolysis, z<0 means at least one missed cleavage. 
Therefore, z-values contain important analytical information. The z-value of any peptide 
(or indeed, any molecule containing only C, H, N and O) can be estimated (e.g. by 
means of mass spectrometry) its distance from the Line, and determined exactly by 
adding/subtracting the masses of zH2Ox until the summed mass fits the Line. The z value 
represents a class of elemental compositions and thus it is more easily determined than 
the elemental composition itself. Such determination requires 1-2 ppm mass accuracy in 
both Mav and Mmono irregardless of the molecular mass. 
 The reason for the focusing of the molecules with z=0 into the Line is the 
peculiar relationship between the isotopic shifts and mass defects of the elements C, H, 
N and O. Indeed, 
 NMD = 1000·(Dc·c + Dh·h + Dn·n + Do·o)/(12·c + h + 14·n + 16·o) (6a) 
and 
 NIS = 1000·(Ic·c + Ih·h + In·n + Io·o)/(12·c + h + 14·n + 16·o), (6b) 
where Dx and Ix are atomic mass defects and elements’ isotopic shifts, respectively.  
Combination of (3) and (4) is equivalent to (Dc=0 in the scale of 12C=12.000): 
c(Ic + 2Ih – 2aDh – 14b*) + n(In – 13b* – Ih – aDn + aDh) + o(Io – aDo – 16b*) = 0, (7)  
where b* = b/1000.  
Since c, o and n are arbitrary numbers, (7) is equivalent to the system of three equations: 
5 
 Ic + 2Ih – 2aDh – 14b* = 0      (8a) 
 In – 13b* – Ih – aDn + aDh = 0     (8b) 
 Io – aDo – 16b* = 0       (8c) 
Using the NIS values3 Dh = 0.007825032, Dn = 0.0030740053, Ic = 0.001078, Ih = 
0.00012197, and In = 0.003646, obtain from (8a-b): a = 0.3481012, b = 0.3982968 ‰.  
Note that (8c) is independent from (8a-b) and contains only values related to oxygen. 
Knowing that Do = -0.005085378 and using the values of a and b from (8a-b), find the 
‘resonance’ value of #Io = aDo + 16b* = 0.004603.  When this value is used, the Line 
becomes a mathematically ideal line, and the central peak in Figure 2 becomes infinitely 
thin.  
The table value of Io is 0.004515, i.e. just 19.5‰ off the resonance value.  Since 
most of the isotopic shift of oxygen comes from 18O, the resonance and table 
abundances of this isotope differ by less than 10‰. Such a difference is within the range 
of natural variations: e.g. oxygen in ambient air is enriched by 23.5‰.4 Instead of Io, the 
value of In could be adjusted downwards by 2% to achieve the resonance, which is also 
within the natural isotopic abundance variation. Alternatively, Ic could be lowered by 
22% or Ih increased by 60%. 
The linear correlation between the isotopic abundances of C, H, N and O and 
their respective monoisotopic mass defects for molecules with z=0 holds true 
irrespective of the mass standard.  Changing the Io value in the range from 0.001000 to 
0.010000 produces a plurality of resonance Io values that create linear features on a 2D 
mass map, but no feature is comparable in size and prominence (number of participating 
peptides) with the one in Figure 1. Thus the a priori probability for the Line to emerge 
by pure chance is less than (0.004603 -0.004515)/(0.010000 – 0.001000), i.e. < 0.01. 
6 
Not all amino acids are proportionally represented in on-Line peptides. As 
expected, amino acids with z = 0 (Table 1) are overrepresented, while sulfur-containing 
residues and aromatic residues Tyr, Phe and Trp that have large positive z-values are 
practically absent. Trifonov has reviewed a bulk of origin-of-life hypothesis and 
determined the consensus order of amino acid emergence.2 We found an overall 
correlation (p=0.05) between the consensus ranking of a residue and the probability to 
be found in on-Line peptides (Figure 4). The correlation means that the more ancient an 
amino acid is, the more likely it is found in on-Line peptides.  
On average, the nine amino acids with z=0 (Table 1) are overrepresented in on-
Line peptides by 23%. Their average consensus ranking is 8.3, as opposed to 11.6 of the 
other nine non-sulphur residues. Even today, the z=0 residues account for 57% of the 
amino acid abundance in natural proteins. When life first emerged, the relative 
abundance of these and other low-z amino acids was certainly higher, possibly as high 
as 100%. In the course of evolution, as was discovered by Zuckerkandl5 et al. and 
recently by Jordan et al.6, the ‘ancient’ amino acids which tend to have low z values 
were consistently lost, being replaced by ‘novel’ amino acids with high z-values. But 
even today peptides with small z comprise the majority of tryptic sequences (Figure 3). 
The ancient polypeptides must be much more concentrated on and around the Line. 
Therefore, the Line is a relic of the time when life was emerging on Earth and was 
employing a limited set of ancient amino acids.  
One of the questions intimately related to the origin of life is why out of the 500 
different amino acids abiotically produced in the seminal experimental series started by 
Miller,7 only ten (G, A, V, L, I, S, T, P, D, and E) are found among the 20 amino acids 
common in terrestrial biological systems.2 Note that out of these ten primary amino 
7 
acids, the first seven have z=0 as residues, and the remaining three have z=0 in the free 
form (i.e. upon water addition). Ribose C5H10O5 that is the basis of RNA and has 
plausible prebiotic syntheses, also has z=0. Thus there may also be, besides empirical, 
also a causal link between having z=0 and being selected as a basis of terrestrial life. To 
reveal this link, one has to find a plausible explanation of the benefit of a linear 
correlation between the mass defects and isotopic shifts of the ‘lucky’ molecules. 
A useful analogue in this case is the ‘slope = 1’ line in mass-independent 
fractionation (MIF) reactions, in which the degrees of enrichment/depletion of several 
isotopes of the same element (e.g. oxygen or sulfur) are independent upon the isotopic 
masses.8  The ‘slope =1’ means a significant complexity reduction in the reacting 
system, as the isotopic masses disappear from the kinetic equation. Gao and Marcus 
have explained MIF by quantum-mechanical effects involving a reduction in the 
number of quantum-mechanical states due to the symmetry of some isotopomers.9  
Similarly, in our case the peptides with z=0 are characterized by a significant 
complexity reduction and thus by a decreased number of quantum-mechanical states. 
Indeed, while in general Mav is defined by 14 parameters (four monoisotopic masses 
and five masses of isotopes and their five relative abundances), the presence of a linear 
correlation between Mav and Mmono reduces the number of parameters to just six (four 
monoisotopic masses and two coefficients of the linear equation).  Therefore, in analogy 
with MIF phenomena where ‘slope=1’ reactions have much higher rates than 
conventional equilibrium fractionation reactions, the presence of the Line must 
accelerate certain reactions involving molecules with z = 0.  MIF is usually observed in 
non-equilibrium processes involving photo-,  electronic excitation or high 
8 
temperatures;8 abiotic synthesis of amino acids and other building bocks of life is 
thought to be involving similar mechanisms.10 
Thus one can hypothesize that the choice of amino acids for terrestrial life has 
been affected by the isotopic abundances of biogenic elements C, H, N, and O: amino 
acids and peptides with z=0 where preferred. Note that within the solar system, isotopic 
abundance of biogenic elements are similar to terrestrial values, while for objects 
originating from the outside space (e.g. some comets), these values may differ by a 
factor of two and more.11  For instance, the ratio 12C/13C is 92 on Earth and ≈20 in the 
Galactic centre.12 In general, terrestrial isotopic abundances are atypical for our 
Galaxy.12 Thus if the feature in Figure 1 is not spurious, life has emerged either in the 
Solar system or in an environment with very similar to terrestrial isotopic abundances of 
biogenic elements. 
  
9 
References 
1. Demirev, P. A. & Zubarev, R. A. Probing Combinatorial Library Diversity by Mass 
Spectrometry. Anal. Chem. 69, 2893-2900 (1997). 
2. Trifonov E. N. The Triplet Code from First Principles, J. Biomolec. Struct. 
Dynamics, 22, 1-11 (2004). 
3. http://physics.nist.gov/cgi-
bin/Compositions/stand_alone.pl?ele=&ascii=html&isotype=some, downloaded 
07/29/2008.  
4. Kroopnick, P. & Craig, H. Atmospheric Oxygen; Isotopic Composition and 
Solubility Fractionation. Science 175, 54–55 (1972). 
5. Zuckerkandl, E., Derancourt, J., Vogel, H. Mutational trends and randomprocesses in 
the evolution of informational macromolecules. J. Mol. Biol. 59, 473-490 (1971). 
6. Jordan, I. K., Kondrashov, F. A., Adzhubei, I. A., Wolf ,Y. I., Koonin, E. V., 
Kondrashov, A. S., Sunyaev, S. A universal trend of amino acid gain and lossin protein 
evolution. Nature 433, 633-638 (2005). 
7. Miller, S. L. A Production of Amino Acids Under Possible Primitive Earth 
Conditions, Science 117, 528 - 529 (1953). 
8. Thiemens, M. H. & Heidenreich, J. E. III. The mass-independent fractionation of 
oxygen: a novel isotope effect and its possible cosmochemical implications. Science 
219, 1073-1075 (1983). 
9. Gao, Y. Q. & Marcus, R. A. Strange and Unconventional Isotope Effects in Ozone 
Formation, Science, 293, 259 – 263 (2001). 
10. Bada, J. L. How life began on Earth: a status report. Earth and Planetary Sci. Lett. 
226, 1-15 (2004). 
10 
11. Wasserburg, G. J., Busso, M., Gallino, R., Nollett, K. M. Short-lived nuclei in the 
early Solar System: Possible AGB sources, Nucl. Phys. A 777, 5–69 (2006).  
12. Wielen, R. & Wilson, T. L. The evolution of the C, N, and O isotope ratios from an 
improved comparison of the interstellar medium with the Sun, Astron. Astrophys. 326, 
139–142 (1997).  
 
11 
Figure legends 
Figure 1. Map of theoretical mass values of 3600 tryptic peptides from mouse 
kidney identified in a shotgun proteomics experiment. 
Figure 2. Distances in ‰ of mass dots in Figure 1 to the line feature. The 
distances are calculated as d = (NIS - a⋅NMD – b)/(1 + a2)0.5. 
Figure 3. Distances in ‰ from the line feature for peptide brutto-formulae, and 
with added or subtracted water molecule.  
Figure 4. Correlation between the degree of enrichment of amino acids in on-
Line peptides and their consensus average rank of emergence in ref. 2. The 
enrichment degree is calculated as (NL/NT)⋅(PT/PL), where NL is the number of 
amino acids of a particular type in on-Line peptides, NT is the number of these 
amino acids in all peptides, PT is the total number of amino acids in all peptides 
and PL is the total number of amino acids in on-Line peptides. 
12 
  
Table 1. Sulfur-free amino acid residues, their z values  
z = c – (n + h)/2, and the consensus order of emergence2 
Alanine, C3H5NO 0 4.0 
Arginine, C6H12N4O -2 11.0 
Asparagine, C4H6N2O2 1 11.3 
Aspartic Acid, C4H5NO3 1 6.0 
Glutamic Acid, C5H7NO3 1 8.1 
Glutamine, C5H8N2O2 0 11.4 
Glycine, C2H3NO 0 3.5 
Histidine, C6H7N3O 1 13.0 
Isoleucine, Leucine C6H11NO 0 11.4, 9.9 
Lysine, C6H12N2O -1 13.3 
Phenylalanine, C9H9NO 4 14.2 
Proline, C5H7NO 1 7.3 
Serine, C3H5NO2 0 7.6 
Threonine, C4H7NO2 0 9.4 
Tryptophan, C11H10N2O 5 16.5 
Tyrosine, C9H9NO 4 15.2 
Valine, C5H9NO 0 
6.3 
 
13 
 
 
 
Figure 1. 
14 
 
 
Figure 2. 
15 
 
 
Figure 3. 
16 
 
 
Figure 4. 


A Production of Amino Acids Under Possible Primitive Earth Conditions,

A universal trend of amino acid gain and lossin protein evolution.

Atmospheric Oxygen; Isotopic Composition and Solubility Fractionation.

How life began on Earth: a status report.

Mutational trends and randomprocesses in the evolution of informational macromolecules.

Probing Combinatorial Library Diversity by Mass Spectrometry.

Short-lived nuclei in the early Solar System: Possible AGB sources,

Strange and Unconventional Isotope Effects in Ozone Formation,

The evolution of the C, N, and O isotope ratios from an improved comparison of the interstellar medium with the Sun,

The mass-independent fractionation of oxygen: a novel isotope effect and its possible cosmochemical implications.

The Triplet Code from First Principles,

Early Life Relict Feature in Peptide Mass Distribution

http://precedings.nature.com/documents/2402/version/1

Early Life Relict Feature in Peptide Mass Distribution

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