Download SUPPLEMENTARY DISCUSSION The applied Hi3 approach relies

SUPPLEMENTARY DISCUSSION
The applied Hi3 approach relies mainly on the assumption that the average intensity
of the three most abundant peptides correlates with the abundance of the
corresponding protein [1]. However, the ionization properties of specific peptides may
influence the corresponding signal intensity during the mass spectrometric
measurement. This may interfere with subsequent calculations of the absolute
quantity of proteins. Moreover, a critical evaluation of the data provided by Silva et al.
[1] suggest that the reproducibility of the quantification might be lower in proteins with
low abundance and small size.
In order to validate the absolute quantification of proteins, Silva et al. [1]
determined the stoichiometry of Escherichia coli proteins belonging to selected wellcharacterized protein complexes. Accordingly, we calculated the stoichiometry of
representative Pseudomonas aeruginosa protein complexes (i.e. ATP synthase F1
and soluble part of the succinate dehydrogenase) as well as of the chaperonins
GroEL and GroES (Tab. D1) based on the Hi3-quantification. Our results correlate
with the stoichiometry of corresponding and well-characterized E. coli protein
complexes / proteins from the literature [2, 3, 4] providing strong evidence that our
quantification is valid.
According to Silva et al. [1] the quantification of low abundant or small proteins
might be less precise. In order to test whether these limitations also apply to our
dataset, the reproducibility of quantitative values of biological replicates was related
to the protein amount and size. Indeed, low abundant proteins (< 4 fmol/µg) exhibited
on average a RSD of 21% whereas the RSD of more abundant proteins (> 4 fmol/µg)
was significantly lower (13% on average, Fig. D1). Therefore, we decided to exclude
lower abundant proteins (< 4 fmol/µg) from our analysis in order to increase
robustness and validity of the presented dataset. Interestingly, the quantification of
proteins shorter than 100 amino acids (RSD: 14%) was found to be as reproducible
as the quantification of proteins longer than 100 amino acids (RSD: 13%) (Fig. D2).
Table D1: Stoichiometry of protein complexes / proteins selected from the P. aeruginosa
PAO1 dataset. Stoichiometry calculated employing absolute quantitative data was compared
to published stoichiometric data of these protein complexes / proteins in E. coli.
Protein complex/
proteins
Sub-units
E. coli
(reference)
P. aeruginosa PAO1
(this study)
α:β:γ:δ:ε
3 : 3 : 1 : 1 : 1 (2)
3 : 3.4 : 0.7 : 1.8 : 2.8
Chaperonin GroEL
& GroES
L:S
2 : 1 (3)
2 : 1.5
Soluble succinate
dehydrogenase
complex
A:B
1 : 1 (4)
1 : 1.4
ATP synthase F1
complex
Figure D1: Reproducibility of protein quantification based on the Hi3-approach is dependent
on the protein concentration. Biological replicates of P. aeruginosa PAO1 were used for
analysis.
Figure D2: Reproducibility of protein quantification seems independent of protein size.
Proteins longer than 100 amino acids (A) can be quantified as reproducible as proteins
shorter than 100 amino acids (B). Biological replicates of P. aeruginosa PAO1 were used for
analysis.
References
[1] Silva JC, Gorenstein MV, Li GZ, Vissers JP, Geromanos SJ. Absolute
quantification of proteins by LCMSE: a virtue of parallel MS acquisition. Mol Cell
Proteomics 2006, 5, 144-156.
[2] Deckers-Hebestreit G, Altendorf K. The F0F1-type ATP synthases of bacteria:
structure and function of the F0 complex. Annu Rev Microbiol 1996, 50, 791-824.
[3] Braig K, Otwinowski Z, Hegde R, Boisvert DC, Joachimiak A, Horwich AL, Sigler
PB. The crystal structure of the bacterial chaperonin GroEL at 2.8 A. Nature 1994,
371, 578-586.
[4] Cecchini G, Schröder I, Gunsalus RP, Maklashina E. Succinate dehydrogenase
and fumarate reductase from Escherichia coli. Biochim Biophys Acta 2002, 1553,
140-157.