Supporting Information
Development of fast photochemical oxidation of protein (FPOP) platform
Ying Zhang, Don L. Rempel, Hao Zhang, and Michael L. Gross*
Experimental
Fitting and Simulation
We fit the mass spectrum by using a custom-built MathCAD-based (version 14.0 M010 by Parametric
Technology Corporation, Needham, MA) fitting program. The basis for the program was previously
described[1] where, in a single stage, a linear combination of isotopic patterns computed from modified
protein molecular formulae are fitted to a peak intensity list derived from the experimental spectrum.
Each modified protein molecular formula corresponds to a component in the spectrum that arises from a
chemical modification of the protein. These modifications included those that existed before and after
the FPOP chemistry and are tabulated in a “modification list” of molecular formulae. We now have
incorporated an additional stage, currently the first stage, that is the construction of a “template” that
models the mass spectrum of the non-“FPOPed” control sample. The role of the template is to account
for any pre-existing modifications to the protein. We modeled each component of the protein spectrum
that was seen in the absence of FPOP by using the protein’s molecular formula and a molecular formula
taken from a “non-FPOP” modification list. This list includes common modifications that are not FPOPintroduced, for example, Na and K. The intensity of the isotopic pattern for each component is scaled so
that the superposition of isotopic patterns fits the peak intensity list of the control mass spectrum. The
template, then, is just the relative intensities in the control spectrum of the components described in the
non-FPOP modification list and their associated molecular formulae. In the second stage, mass spectra
of the protein following FPOP are analyzed. Each molecular formula from the “FPOP” modification list
results in a separate modified template. The molecular formula of each component represented in the
modified template is just the corresponding molecular formula from the original template with the FPOP
modification molecular formula added on. The relative intensities of the peaks for components
represented in the modified template remain the same as in the original template. For example, the
unmodified template becomes the model for the non-FPOP components in the FPOP spectra. We then
fit the peak intensity list derived from the experimental FPOP spectra by scaling the intensity of the
peak list arising from each modified template and superimposing them. The fraction of unmodified
species (% unmod) is the ratio of unmodified template intensity to the total of the unmodified and
modified template intensities resulting from the fit. The outcome, “% unmod”, provides a quantitative
way to compare quickly the FPOP results obtained under different conditions without doing digestion
and bottom-up proteomics analysis. All the results reported here were obtained by this template-fitting
process to afford “% unmod”. For the protein under study.
References
1. Gau, B.C., Sharp, J.S., Rempel, D.L., Gross, M.L.: Fast photochemical oxidation of protein footprints faster
than protein unfolding. Anal Chem 81(16), 6563-6571 (2009). doi:10.1021/ac901054w
Experiments
#2
#3
A
#4
Int
#1
B
C
m/z
% unmodified protein for standard experiments
Condition
Exp. #1
Exp. #2
Exp. #3
Exp. #4
Average
Non-laser (A)
58.2
56.7
58.0
58.9
58.0
0.9
Non-H2O2 (B)
59.6
60.1
60.1
59.8
59.9
0.2
FPOP (C)
25.1
24.2
24.7
25.1
24.8
0.4
Figure S1. Mass spectra of FPOP-labeled cyt. c (+9) obtained by using a modified FPOP platform in
quadruplicate for (A) “non-laser” control, (B) “non-H2O2” control, and (C) usual FPOP conditions. The % unmod
for each experiment is included in the table accompanying the figure.
Experiments
#2
#3
A
#4
Int
#1
B
C
m/z
% unmodified protein for standard experiments
Condition
Exp. #1
Exp. #2
Exp. #3
Exp. #4
Average
Non-laser (A)
57.4
56.7
57.6
59.0
57.7
1.0
Non-H2O2 (B)
63.8
64.4
64.1
64.6
64.2
0.4
FPOP (C)
25.3
23.5
24.9
24.0
24.4
0.8
Figure S2. Mass spectra of FPOP-labeled cyt. c (+12) obtained by using a modified FPOP platform in
quadruplicate for (A) “non-laser” control, (B) “non-H2O2” control, and (C) usual FPOP conditions. The % unmod
for each experiment is included in the table accompanying the figure.
Experiments
#2
#3
A
#4
Int
#1
B
C
m/z
% unmodified protein for standard experiments
Condition
Exp. #1
Exp. #2
Exp. #3
Exp. #4
Average
Non-laser (A)
60.7
59.1
58.8
60.5
59.8
1.0
Non-H2O2 (B)
63.0
65.3
64.0
64.4
64.2
1.0
FPOP (C)
27.0
26.0
26.4
26.5
26.5
0.4
Figure S3. Deconvoluted mass spectra of FPOP-labeled cyt. c obtained by using a modified FPOP platform in
quadruplicate for (A) “non-laser” control, (B) “non-H2O2” control, and (C) usual FPOP conditions.
Deconvolution is done by MagTran. The % unmod for each experiment is included in the table accompanying the
figure.
#4
Int
#1
Experiments
#2
#3
m/z
% unmodified protein for experiments using different buffer
[Buffer] and
[H2O2]
10 mM PBS
15 mM H2O2
25 mM Tris
15 mM H2O2
25 mM Tris
30 mM H2O2
10 mM Tris
30 mM H2O2
% unmod
26.3
46.3
37.4
27.8
Figure S4. Mass spectra of FPOP-labeled cyt. c in different buffer systems. The % unmod for each experiment is
included in the table accompanying the figure.