Supporting Information Note 1:
Preparation of TMPP and the detail procedures for synthesis of each compound.
Preparation of TMPP
13
C-dimethyl sulfate was used to synthesize H-TMPP (13C9-TMPP); this approach
yields nine 13C-labeled carbons. All chemicals were analytical grade.
The detail procedures for synthesis of each compound as follows (Figure S1):
1) Synthesis of compound 1
A round-bottle flask containing 1.92 g m-trihydroxybenzene, 10.2 g potassium
carbonate anhydrate, and 50 mL dry acetone was heated and refluxed. 13C3-labeled
dimethyl sulfate (6.5 g) was slowly added into the system, using a syringe, every 15
min. After the solution was refluxed for 5 h, the reaction was allowed to cool down,
and was then quenched by adding 18 mL H2O and 2 mL ammonium into the system,
while stirring for 1 h at room temperature. The solution was then extracted with 200
mL diethyl ether, and the organic layer was then washed using diluted hydrochloric
acid, diluted sodium hydroxide solution, and saturated salt solution. Subsequently, the
organic layer was dried using magnesium sulfate. After removing the organic solvent,
and purifying the residual by column chromatography using petroleum ether and ethyl
acetate as eluent, 1.2 g product was obtained with a yield of 46%.
2)
Synthesis of compound 2
Compound 2 was synthesized in a round-bottle flask by refluxing a solution of 0.785
g compound 1, 0.25 g zinc chloride anhydrate, and 0.8 mL distilled phosphorus
trichloride under anhydrous and anaerobic conditions for 11 h. Thereafter, the solution
1
was diluted with toluene, and the insoluble solid was washed with toluene several
times. The toluene was then removed using a pipette. Ammonium (10 mL) of was
subsequently added to the residue, and the system was extracted using 50 mL toluene.
The organic layer was then washed with saturated salt solution and dried using
magnesium sulfate. After the solvent was removed, the residual was recrystallized
from the ethanol/diethyl ether mixture, and 0.577 g solid was obtained, with a yield of
70%.
3) Synthesis of compound 3
In an ice bath, 0.76 mL bromoacetyl bromide in 10 mL dichloromethane was added
slowly into a reaction flask containing 0.71 mL pyridine and 10 mL dry
dichloromethane. Then, 0.6 g N-hydroxysuccinimide was added into the solution,
which was then continuously stirred for 1 h. The solution was subsequently washed
with saturated salt solution and dried using magnesium sulfate. The solvent was
removed, and the residual recrystallized using a mixture of dichloromethane and
petroleum ether; 1.12 g solid product was obtained, with a yield of 91%.
4) Synthesis of 13C9-TMPP-Ac-Osu
Compound 3 (0.228 g) was dissolved in 7 mL of toluene, and compound 2 (0.523 g)
was dissolved in 10 mL of toluene. When the two toluene solutions were mixed
together, a solid appeared immediately. Petroleum ether (30 mL) was added to this,
and the solution was stirred for 1 h. The product was filtered and washed with
petroleum ether. After the solid was dried under vacuum, 0.756 g product was
obtained, with a yield of 100%.
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The structures of the intermediate compounds and of
13
C9-TMPP were
characterized by proton nuclear magnetic resonance (1H NMR) spectroscopy (Bruker,
Billerica, MA, USA). We applied Fourier-transform MS (FTMS, Bruker Apex IV) to
accurately measure the molecular weight of 12C-TMPP and 13C9-TMPP (Figure S2).
Figure legend:
Figure S1. A. The synthetic route of 13C9-TMPP-Ac-OSu.
1) Compound 1 was synthesized by the methylation reaction of m-trihydroxybenzene
and dimethyl sulfate using potassium carbonate anhydrate as catalyst under reflux
in dry acetone. During the reaction, every 10-15 min, one fourth amount of the
total 13C3 -labeled dimethyl sulfate was added into the system.
2) Using zinc chloride anhydrate as catalyst, compound 2 was synthesized by reflux
the solution of compound 1 and distilled phosphorus trichloride in anhydrous and
anaerobic conditions.
3) In ice bath of dichloromethane, compound 3 was synthesized by the bromination
reaction of bromoacetyl bromide and N-hydroxysuccinimide in pyridine as
solvent.
4) The target compound (13C9-TMPP-Ac-OSu) was synthesized by the reaction of
compound 2 and compound 3.
B. Using the information provided by 1H NMR and FT mass spectrum, the structure
of synthetic TMPP was determined. This 1H NMR spectrum corresponded to that of
3
TMPP. The 1H NMR spectrum of this synthetic TMPP and the chemical shifts were as
follows: (400 MHz, CD3CN): 6.23 (d, J = 5.08 Hz, 6H), 4.60 (d, J = 14.76 Hz, 2H),
3.86 (d, J = 145.36 Hz, 9H), 3.61 (d, J = 146.04 Hz, 18H), 2.73 (s, 4H). The 1H NMR
spectrum of synthetic TMPP and the corresponding hydrogen sites are shown in
different colors.
C. The molecular weights of synthetic TMPP were measured by FT mass spectrum,
and corresponded to 12C-TMPP (MW = 688.215 Da) and 13C9-TMPP (MW = 697.246
Da), respectively.
D. TMPP has high derivatization efficiency using on-tip derivatization approach.
TMPPP derivatization efficiency grows high as mole ratio (reagent: peptides)
increases. The derivatization efficiency reaches above 90% when the weight ratio is
more than 16:1. We used 20:1 ratio in this study for high labeling efficiency.
Figure S2. Isotope TMPP as a labeling reagent is accurate and efficient in the
quantification of α-casein. Light and heavy-TMPP labeled peptides from α-casein and
were mixed at 10:1(L:H), 5:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:5, and 1:10 ratio. The
MS-spectrum of peptide EGIHAQQK (mass = 909.467 Da, z =2) showed that the two
isotopic peaks were separated by 9 Da and the ratios of L/H were close to the
expected abundance ratios in all groups. Because of the addition of TMPP moiety on
N-termini of peptides, the m/z of
12
C-TMPP labeled peptide is 741.83 and m/z of
13
C9-TMPP labeled peptide is 746.33. The corresponding TIC figures of each group
are shown in the left part.
Figure S3. The scatter plot of peptide ratios shows the accuracy and variability of
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quantification. Light and heavy-TMPP labeled peptides were mixed at 10:1(L:H), 5:1,
3:1, 2:1, 1:1, 1:2, 1:3, 1:5, and 1:10 ratio. The median ratios also concentrated closely
to the expected abundance ratios, but peptide ratios have much higher CV, even
reaching 65%. ＋ points the mean ratio of each group. The linear regression analysis
of observed median peptide ratios compared with expected ratios were logarithmically
plotted. The linear regression and 99% prediction intervals lines are shown in the
figure. The observed and expected ratios are linearity correlated and R2=0.9967,
p-value < 0.0001.
Figure S4. Volcano plots represent the p-values versus peptide ratio changes of L/HTMPP peptides mixed at different ratios. The dash line shows the median ratio of each
group.
Figure S5. A statistical analysis of N-terminal amino acid in the indentified peptides
from 1:1 mixed group. Volcano plot representing the p-values versus ratio changes of
each kind of N-terminal amino acid shows the ratio distribution of peptides. The
abbreviations of each amino acid were shown at the top. The dash lines show a
significant ratio change (p-value < 0.001, ratio change > 1.5).
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