Labeling and Quantitation problem set answers
1. (A) Reversed-phase LC separation is based on differences in hydrophobicity of an
analyte (most hydrophobic elutes last). More hydrophobic species will have more sites of
interaction with a hydrophobic stationary phase (typically C18, for peptides). This
interaction is based on Van der Waals forces. The mechanism of isotopic separation is based
on the kinetic isotope effect, which is a dependence of the rate of a chemical reaction on
the isotopic identity of an atom in a reactant. Isotopic effect will change rate of
hydrophobic interactions of an analyte with the stationary phase, as well as the magnitude
of Van der Waals forces.
Isotopic rate changes are most pronounced when the relative mass change is greatest since
the effect is related to vibrational frequencies of the affected bonds. For instance, changing
a hydrogen atom to deuterium represents a 100% increase in mass, whereas in replacing
carbon-12 with carbon-13, the mass increases by only 8%. The rate of a reaction involving a
C-H bond is typically 6 to 10 times faster than the corresponding C-D bond, whereas a 12C
reaction is only ~1.04 times faster than the corresponding 13C reaction (even though, in
both cases, the isotope is one atomic mass unit heavier). More detailed explanation of the
kinetic isotope effect can be found here:
http://en.wikipedia.org/wiki/Kinetic_isotope_effect , or elsewhere.
(B)
It would be a reasonable assumption that the more D atoms are in a molecule, the
more it will change its RPLC elution time. Also, the isotopic effect would have more impact if
the D's are situated in hydrophobic part of the molecule, which is interacting with the
stationary phase, whether if the D's were situated in a more hydrophilic part, their effect
might have been negligible.
Therefore, the H3/D3 forms of N-acetoxysuccinimide will have the lowest degree of isotopic
separation, and H8/D8 forms of the commercial ICAT reagent – the highest degree. The
experimental evidence of that can be found in the paper:
Fractionation of Isotopically Labeled Peptides in Quantitative Proteomics. R. Zhang, C. S.
Sioma, S. Wang, and F. E. Regnier; Anal. Chem., 2001, 73 (21), pp 5142–5149
2. The advantages of chemical labelling before digestion: (a) the label is introduced earlier in
the analysis flowchart, thus lessening uncompensated quantitation errors; (b) sample
cleanup after labelling (which is where the sample becomes the most dirty) is easier, as the
difference in size and other properties between a small chemical labelling reagent and a
protein is more pronounced than between the reagent and a peptide.
The advantage of chemical labelling after digestion: labelling can be more efficient, as some
of the reacting sites may be hidden inside the protein core, and less accessible for the
reagent. The reacting sites on peptides are more readily available.
3. Exact masses of the four isotopic versions of the iTRAQ reagent are:
Reporter ion mass Reporter ion
Balance group
Total tag exact
(nominal), Da
exact mass, Da
exact mass, Da
mass, Da
114
114.1110
31.0026
145.1136
115
115.1144
29.9992
145.1136
116
116.1114
28.9983
145.1097
117
117.1148
27.9992
145.1140
Interestingly, the masses of the 2 of 4 tags are isobaric up to 5th digit.
∆ M between the neighbouring peaks are: 0.0036 and 0.0004 Da.
Resolving power which would be necessary for baseline resolution of the iTRAQ-labelled peptides
(added mass 145, assuming that only N-terminus was labelled) with masses 1000 Da and 2500 Da
(assuming singly-charged peptides):
Peptide
mass\Resolving
power
1000
2500
∆ M = 0.0036 Da
∆ M = 0.0004 Da
1145/0.0036 =
1145/0.0004 =
318,055
2645/0.0036 =
734,722
2,860,000
2645/0.0004 =
6,612,500
Therefore, the possibility of baseline resolution of the iTRAQ-labelled peptides in the MS mode
varies. With the currently available MS instrumentation, it would not be possible to resolve the first
two peaks (corresponding to reporter ion masses 114/115 and 115), as this would require resolving
power in the range of 10 to 100 mln. Resolution of the peaks corresponding to reporter ion masses
114/115 and 117 (∆ M = 0.0004 Da) is hardly possible, too. Yet, resolution of the peaks
corresponding to reporter ion masses 114/115 and 116 (∆ M = 0.0036 Da) is possible by modern
FTICR instruments (typically >50000 broadband, >1,000,000 narrowband). With all other types of
detectors, the peptides labeled with all 4 tags will yield a single peak in the MS mode.
4. The mass defect introduced by the MDL is -0.1634 Da (if a peptide contains 1 Cys residue)
and -0.3268 Da (for peptides containing 2 cysteines).
Mass defect introduced by a single phosphate group (HPO3) is -0.0277 Da.
To estimate the mass defect of a peptide with M = 1500 Da, one can consider an
“averagine” amino acid, i.e. an “average” amino acid which composition was calculated
based on the statistical occurrence of all amino acids in proteins from RIP protein data base
(M.W. Senko, S. C. Beu and F.W. McLafferty; Determination of monoisotopic masses and ion
populations for large biomolecules from resolved isotopic distributions. J Am Soc MS 1995 pp.
229-233).
The elemental composition used for averagine was found to be:
C 4.9384
H 7.7583
N 1.3577
O 1.4773
S 0.0417,
and its exact mass (monoisotopic) is 111.0541 Da.
Therefore, mass defect for an “average” AA with the nominal mass of 111 Da is 0.0541 Da,
and that for an “average” peptide with the nominal mass of 1500 Da will be 0.731 Da.
Therefore, labelling of an 1-Cys-containing peptide with MDL will result in 0.1634 Da shift to
the left, yielding mass defect of 0.5676 Da, and adding a 1 phospho group will result in the
mass defect of 0.7033 Da. Clearly, in the second case the phosphopeptide mass region will
overlap to large extent with the mass region of peptides not containing phospho groups (see
the figure below, reference: Probabilistic Enrichment of Phosphopeptides by Their Mass
Defect, C.Bruce, M.A. Shifman, P.Miller, E.E. Gulcicek; Anal. Chem., 2006, 78 (13), pp 4374–
4382). The overlap will decrease in the case of doubly- and triply-phosphorylated peptides.
Yet, the mass defect shift brought by a phospho group may assist in peptide ID based solely
on exact mass values (“Utility of Accurate Mass Tags for Proteome-Wide Protein
Identification” T. P. Conrads, G.A. Anderson, T.D. Veenstra, L. Paša-Tolić, and R.D. Smith
Anal. Chem., 2000, 72 (14), pp 3349–3354).
Distribution of peptide numbers and phosphorylation probability for tryptic peptides
with zero, one, two, and three phosphates and in the mass range 1200−1201 Da.
The probability of phosphorylation is (N1 + N2 + N3)/(N0 + N1 + N2 + N3), where N0, N1,
N2, and N3 are the number of peptides with zero, one, two, and three phosphates,
respectively, within each bin of the histogram. Each bin size is 0.01 Da.