Identification of Protein Thiazolidination as a Novel Molecular Signature for Oxidative Stress and Formaldehyde Exposure

Article abstract of DOI:10.1021/acs.chemrestox.6b00271

Chemical modifications of proteins have been well-documented to play important roles in normal cell physiology such as cell signaling and protein functions. They have also been demonstrated to be one of the milestones in the pathophysiology of many human diseases such as cancer, age-related pathology, and neurodegenerative disorders. Here, we report the initial identification of a novel protein modification, cysteine thiazolidination, through reaction with endogenous and exogenous formaldehyde with cysteine residues in proteins. Using an isotope-dilution liquid chromatography-tandem mass spectrometric (LC-MS³) method, we initiated the study by quantitating thioproline in formaldehyde-treated Escherichia coli (E. coli) protein. The study was then extended to quantitate thioproline in protein obtained from formaldehyde- and oxidant-exposed E. coli. Furthermore, N⁶-formyllysine, a well-defined formylation product between formaldehyde and lysine, was exploited in a comparative study to evaluate the relative reactivity and amount of cysteine thiazolidination in the reaction of formaldehyde with proteins. It is anticipated that cysteine thiazolidination may serve as a novel biomarker for oxidative stress and formaldehyde exposure.

Full text of DOI:10.1021/acs.chemrestox.6b00271

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Article
Identification of Protein Thiazolidination as a Novel Molecular
Signature for Oxidative Stress and Formaldehyde Exposure
Jingjing Liu, K. K. Jason Chan, and Wan Chan
Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.6b00271 • Publication Date (Web): 02 Nov 2016
Downloaded from http://pubs.acs.org on November 4, 2016
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Identification of Protein Thiazolidination as a Novel Molecular
Signature for Oxidative Stress and Formaldehyde Exposure
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Jingjing Liu,† K. K. Jason Chan,‡ and Wan Chan*,†,‡
† Environmental Science Programs and ‡ Department of Chemistry, The Hong Kong
University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong
* Correspondence author
Address: Room 4520, Department of Chemistry, Academic Building, Clear Water Bay,
Kowloon, Hong Kong
Eꢀmail: chanwan@ust.hk, Phone: +852 2358ꢀ7370, Fax: +852ꢀ2358ꢀ1594.
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ABSTRACT
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Chemical modifications of proteins have been wellꢀdocumented to play important
roles in normal cell physiology such as cell signaling and protein functions. They
have also been demonstrated to be one of the milestones in the pathophysiology of
many human diseases such as cancer, ageꢀrelated pathology, and neurodegenerative
disorders. Here we report the initial identification of a novel protein modification,
cysteine thiazolidination, through reaction with endogenous and exogenous
formaldehyde with cysteine residues in proteins. Using an isotopeꢀdilution liquid
chromatographyꢀtandem mass spectrometric (LC−MS3) method, we initiated the
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study by quantitating thioproline in formaldehydeꢀtreated Escherichia coli (E. coli)
protein. The study was then extended to quantitate thioproline in protein obtained
from formaldehydeꢀ and oxidantꢀexposed E. coli. Furthermore, N6ꢀformyllysine, a
wellꢀdefined formylation product between formaldehyde and lysine, was exploited in
a comparative study to evaluate the relative reactivity and amount of cysteine
thiazolidination in the reaction of formaldehyde with proteins. It is anticipated that
cysteine thiazolidination may serve as a novel biomarker for oxidative stress and
formaldehyde exposure.
Keywords: Protein modification, thiazolidination, thioproline, oxidative stress,
LCꢀMS/MS
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INTRODUCTION
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Proteins are molecular vehicles that drive a myriad of cellular processes, and the
diverse biological functions they exhibit are attributed to their specific
substrateꢀfitting shapes as well as unique arrangements of functional groups. Many
cellular proteins contain some form of nonꢀcanonical postꢀtranslational
modifications (PTMs) which can carry physiological significance and their
formations are normally mediated through enzymes. However, the constant
presence of reactive chemical species inside the cell, arising both endogenously and
exogenously, can also lead to the proteins being modified through unintended
chemical reactions. These PTMs could alter protein structures and their
bioactivities, which may lead to adverse effects on cell physiology.1,2 PTMs
commonly involve electrophilic species, which form covalent adducts with
nucleophilic amino acid residues through alkylation, carbonylation, glycation, or
phosphorylation. Correlations between these modifications and their physiological
significance in cell signaling,3,4 functions,5ꢀ8 and pathophysiology9,10 are
wellꢀdocumented.
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Among the many reactive electrophiles known to modify proteins, formaldehyde
that is ubiquitous in both indoor and outdoor air, present in smoked and barbequed
foods and even generated endogenouly from processes such as lipid peroxidation11,
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12 has attracted a lot of attention as a PTM agent, especially since it is also a known
human carcinogen.13 Formaldehyde is known to condense swiftly with lysine and
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arginine residues in peptides to produce N
ꢀformylated products.14ꢀ17 For example,
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the formation of N6ꢀformyllysine has been used as a biomarker to assess the extent
of exposure to oxidative stress (Figure 1).14 The addition of formyl groups to
peptide residues has also been implicated in the misfolding, loss of function, and
hydrolysis of proteins.18ꢀ20 It was also discovered that formylation could result in
cytotoxic protein aggregation products.21, 22
Recently, we reported on the doseꢀdependent formation of
thiazolidineꢀ4ꢀcarboxylic acid (thioproline) when formaldehyde condenses with
cysteine, present as free amino acid in the extraꢀcellular fluid of Escherichia coli (E.
coli) exposed to oxidative stress.23 Since cysteine is an essential building block of
proteins, the aim of this study is to test our hypothesis that formaldehyde would
also react with cysteine residues within a polypeptide to form stable thiazolidine
adducts.
Using a liquid chromatographyꢀcoupled tandem mass spectrometric (LC−MS3)
method with high sensitivity and selectivity, we demonstrated in this study for the
first time that cysteine residues in proteins indeed react with formaldehyde in vitro
through a novel thiazolidination process to form a cyclic thioproline residue in a
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doseꢀdependent manner (Figure 1). This newly identified protein modification was
also observed to occur in bacterial proteins isolated from E. coli whole cells that
were treated with formaldehyde, along with other oxidative stress generators
(Fe2+ꢀEDTA, hydrogen peroxide (H2O2), and sodium hypochlorite (NaOCl)).To
establish the relative frequency of this newly identified cysteine thiazolidination
event against wellꢀestablished formylation events during the reaction between
formaldehyde and proteins, we also performed a comparative study in parallel on
the formation of N6ꢀformyllysine.24,25
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EXPERIMENTAL SECTION
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Chemicals and Reagents. All chemicals and reagents of the highest purity
available were used without further purification unless otherwise stated. ꢀcysteine,
N6ꢀformyllysine,
ꢀthioproline, fluorenylmethyloxycarbonyl chloride (FmocꢀCl),
formaldehyde, formaldehydeꢀ 2, and Streptomyces griseus protease were obtained
from Sigma (St. Louis, MO). Isotope labeled ꢀthioproline and ꢀthioprolineꢀ 2, were
synthesized by reacting formaldehydeꢀ 2 with ꢀcysteine and purified by HPLC as
described previously.23 Fmocꢀ
ꢀthioproline and a thioprolineꢀcontaining peptide
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L
L
d
L
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d
d
L
L
(ThioprolineꢀArgꢀGluꢀGlnꢀArgꢀLeuꢀGlyꢀArgꢀGlnꢀTrpꢀAlaꢀValꢀGlyꢀHisꢀMet) were
acquired from GL Biochem (Shanghai, China). Protein for in vitro experiments was
isolated inꢀhouse from cultured E. coli using the method described below. LC−MS
grade acetonitrile and methanol were purchased from J.T. Baker (Philisburg, NJ).
Deionized water was further purified by a MilliꢀQ Ultrapure Water System (Billerica,
MA) and was used in all the experiments.
Protein Isolation. Whole protein for in vitro experiments and in toxicants
exposed E. coli was isolated using the method described previously.26 In brief, E.coli
cells after being collected by centrifugation were washed three times with potassium
phosphate buffer (100 mM, pH 7.4) and then resupended in 1M Tris buffer containing
20% glycerol (v/v). After ultrasounication and centrifugation (18,000 g, 50 min), the
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supernatant containing cellular protein was collected and added with saturated
ammonium sulfate to precipitate the proteins. Cellular protein was then collected by
centrifugation (18,000 g, 30 min).
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Instrumental Analyses. HPLC analysis and purification were performed on an
1100 series HPLC system equipped with a diode array detector (Agilent, Palo Alto,
CA). Highꢀresolution mass spectrometry (MS) and product ion scan (MS/MS)
experiments were performed on a Xevo G2 QTOF mass spectrometer with a standard
electrospray ionization interface (Waters, Milford, MA). LC–MS/MS and LC–MS3
analyses were performed on an Agilent 1100 HPLCꢀcoupled with an API 4000
QTRAP tandem mass spectrometer with a TurboV ion source (AB Sciex, Foster City,
CA). UV spectrometricꢀbased quantitation of protein was performed on a Varian UV–
vis absorption spectrophotometer (Cary 50, Walnut Creek, CA).
Optimization of Reaction Conditions for the Derivatization of Thioproline
with Fmoc-Cl. To determine the amount of FmocꢀCl that is needed for the
derivatization reaction of thioproline, we varied the reagentꢀtoꢀthioproline molar
ratios by adding different amounts of FmocꢀCl to thioprolineꢀfortified (1 nmole)
hydrolysates of purified protein (100 µg) (50:1, 100:1, 200:1, 500:1, 1000:1, 2000:1,
and 5000:1, FmocꢀCl:thioproline) at sodium borate buffer (150 mM, pH 8.0).27 The
mixtures were left to stand in room temperature for 30 min with occasional
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vortexꢀmixing before being analyzed using LCꢀMS3 as described below to evaluate
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the extent of conversion of thioproline to 1.
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Reaction of Formaldehyde with Purified Protein. Solutions of purified E. coli
protein (100 µL, 1000 µg/mL) in potassium phosphate buffer (100 mM, pH 7.4) were
incubated with formaldehyde with final concentrations ranging from 0.6 to 2.5 mM at
37 oC for 1 hr. The formaldehydeꢀtreated protein samples (100 µg) were then
hydrolyzed with protease (10 µg, 37 oC for >16 hr), as described elsewhere.28
Following the enzymatic digestion of the protein, the enzymes were removed by
ultrafiltration using a 10,000 Da molecular weight cutꢀoff Nanosep Centrifugal
Device (Pall Life Sciences). The filtrates containing both naturally occurring and
formaldehydeꢀmodified amino acids (e.g. thioproline and N6ꢀformyllysine), were
derivatized with FmocꢀCl (Figure 2), with an added deuteriumꢀlabeled internal
standard, before they were analyzed by LCꢀMS3 as described below.
Quantitation of Thioproline in Formaldehyde-Treated and Oxidized E. coli
Protein Extract. E.coli (DH5α, ATCC) cultured to midꢀlog phase were harvested by
centrifugation. The cell pellet was washed twice with PBS, and then reꢀsuspended in
PBS. The resuspended cells were treated with formaldehyde (5ꢀ17 mM final
concentration) and the following oxidants: Fe2+ꢀEDTA (0.5ꢀ2.5 mM), NaOCl (0.1ꢀ1.0
ꢁM), and H2O2 (1.25 ꢀ8.5 ꢁM). After 1 hr of exposure, the cells were collected by
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centrifugation, the cellular protein isolated as described above, quantitated by UV
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spectrometry (280 nm), and digested with protease for LC–
MS3 analysis.
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LC–MS3 Analysis. A 10 ꢁL aliquot of the Fmocꢀderivatized protein hydrolysate
was loaded into a GraceSmart C18 column (150 × 2.1 mm, 5 ꢁm), and eluted at 0.3
mL/min with a gradient of acetonitrile (A) in 0.1% formic acid (v/v) in water (B) to
separate the Fmocꢀtagged amino acids. The gradient elution program started from 20%
B (v/v), programmed linearly to 100% B in 20 min, and held for another 5 min before
reconditioning to 20% B for 5 min.
After diverting the first 8 min of the LC eluate to waste, the effluent containing
was directed to a QTRAP mass spectrometer for MS3 analysis. The electrospray
ionization source was operated in positive ion mode with the following optimized
parameters for voltages and source gas: ion spray voltage, 5500 V, collision energy,
15V, declustering potential, 60 V, and excitation energy, 150 V. The ion source gas I,
gas II, curtain gas, collision gas, and temperature of gas II were set to 30, 40, 25, 5,
and 400 °C, respectively. The mass spectrometer was set to monitor the fragments of
the native (m/z 356→134→88) and isotope labeled (m/z 358→136→90) thioproline.
1
Using a similar LCꢀMS/MS method, the Fmocꢀtagged lysineꢀformaldehyde adduct,
was analyzed in another analysis with the mass spectrometer set to monitor the
2,
fragments of m/z 397→175.
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Calibration. A stock solution of thioproline at 500 ꢁg/mL was prepared in
methanol/water (1:1) and stored at ꢀ20 oC before use. The calibration standard
solutions of thioproline (10, 25, 50, 100, 500,1000 ng/mL), were prepared by serial
dilution of the stock solution, to which was added the isotopeꢀlabeled internal
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standard (Fmocꢀlꢀthioprolineꢀ
described above. Calibration curves were established by plotting the peak area ratios
of to the isotopeꢀlabeled internal standard against the concentrations of thioproline
d2), derivatized with FmocꢀCl, and LC–
MS3 analyzed as
1
in the calibration standard solutions. Using a similar approach, the calibration curve
for N6ꢀformyllysine was also established.
Method Validation. The overall efficiency of the analytical method, which
comprised of the protein isolation, enzymatic digestion and chemical derivatization
steps, was determined using a synthetic thioprolineꢀcontaining peptide. The validation
experiment entailed spiking 100 ꢁg of purified protein with different amounts of the
thioprolineꢀcontaining peptide (300ꢀ3000 pmol), followed by protein isolation,
protease digestion to release thioproline, addition of isotopeꢀlabeled internal standard,
derivatization with FmocꢀCl, and LC–MS3 analysis, as described above. The
efficiency of the method was evaluated by plotting the measured quantities of
thioproline against the quantity of added thioprolineꢀcontaining peptide.
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RESULTS AND DISCUSSION
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Optimization of the Derivatization Condition. FmocꢀCl is a reagent originally
developed for amino group protection and is increasingly being used as a
derivatization reagent for biogenic amines.29,30 It introduces an aromatic Fmoc
carbamate group to amines and increases their hydrophobicity for reversedꢀphase
HPLC (RPꢀHPLC) analysis.31 Previous studies have demonstrated that FmocꢀCl can
react efficiently with amineꢀcontaining amino acids, food contaminants, and
neurotransmitters, in a slightly alkaline medium to produce stable Fmocꢀtagged
derivatives for their analysis by RPꢀHPLC.32
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As the basis for our method, we have previously used FmocꢀCl and ethyl
chloroformate as derivatization agents to derivatize thiazolidines prior to their LC–
MS/MS analysis.23,27 Because the Fmocꢀthioproline derivative formed by reacting
thioproline with FmocꢀCl is more hydrophobic and can be efficiently extracted using
ethyl acetate for LC−MS/MS analysis,27 FmocꢀCl was therefore used as the reagent
for optimizing the chromatographic behavior of thioproline for LC–MS3 analysis
(Figure 2). We have previously demonstrated that FmocꢀCl reacts rapidly with
thioproline at room temperature,23 and in this study, we have further optimized the
amount of reagent needed for the derivatization reaction. By using protein hydrolysate
from hydrolyzing 100 µg of control protein as the reaction medium, we determined
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that a molar ratio of 2000:1 for FmocꢀCl to thioproline was necessary for the efficient
formation of the conjugate (Figure S1), and further increases in the molar ratio of
FmocꢀCl did not improve the yield of the derivative. Thus, the molar ratio of 2000:1
was used for the entire study.
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It is not surprising that such a high molar ratio of FmocꢀCl:thioproline (2000:1)
is needed for the derivatization reaction, because the amount of free thioproline
produced during protein hydrolysis would be around a thousandꢀ fold less than all the
other amino acids also liberated. All these free amino acids would also react with
FmocꢀCl via their amino terminus and compete with thioproline for the derivatization
reagent. Thus, a high molar ratio (2000:1) of FmocꢀCl to thioproline is needed for
efficient conversion of thioproline in a protein hydrolysate to its Fmoc derivative, 1.
Comparative Study of LC–MS/MS and LC–MS3 Methods for Proteinic
Thioproline Determination. LC–MS3 analysis has previously been demonstrated to
be highly specific and being more sensitive than LC–MS/MS for bioanalysis.33,34 To
address the challenge of analyzing
1 in the presence of high concentrations of
interfering amino acids in the protein digest, we compared the performance of the
newly developed LC–MS3 method with that of our previously developed LC–MS/MS
method for analyzing thioproline in protein digests.27 Our analyses revealed that LC–
MS3 analysis provided a similar sensitivity as that of the previously developed LC–
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MS/MS method in which the QTRAP MS was operated in multipleꢀreaction
monitoring (MRM) mode. Specifically, we were able to detect one thioproline in the
presence of 106 amino acids (from 100 µg of protein) using both methods.
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Nevertheless, the analysis revealed the LC–MS3 method provided higher
selectivity for identifying compound
1 in the complex matrix of protein digest than
LC–MS/MS analysis in MRM mode (Figure 3 and S2), where we observed
significantly lower interference in the LC–MS3 analysis of a protein hydrolysate from
100 µg of hydrolyzed protein (Figure S2), which was obtained from E. coli cells
exposed to formaldehyde (8.5 mM). It is also believed that the added transition in the
MS3 analysis would increase the specificity of identifying thioproline in the study. A
similar observation of higher confidence of quantitation was also observed in previous
studies when LC–MS3 was used to analyze oxidatively damaged DNA lesions.33,34
Considering the high selectivity and confidence of LCꢀMS3 for analyzing thioproline
in protein hydrolysate, the LC–MS3 method was adopted to quantify the amount of
thioproline formed in cellular protein.
Method Validation. A thioprolineꢀcontaining peptide was custom synthesized
and used to validate the method. The method validation entails spiking 100 ꢁg of E.
coli protein with 300ꢀ5000 pmol of the peptide followed by protein isolation, protease
hydrolysis, derivatization with FmocꢀCl, and LC–MS3 analysis of the digests, as
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described in the Experimental Section. The yield of thioproline, which corrected for
potential loss of the adduct during the protein isolation and in the digestion process,
together with the loss of signal for incomplete derivatization, was found to be 72.2%
of the theoretical value (Figure S3), which represents the overall efficiency for the
method. Measurement of thioproline was thus corrected by a factor of 1.4 to arrive at
the quantity of thioproline in the isolated protein samples.
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Quantitation of Thioproline in Purified Protein and in Protein in E. coli
Treated with Formaldehyde. The validated method was applied to quantitate
thioproline as a novel modification to cysteine both in cellꢀfree protein exposed to
formaldehyde and in protein isolated from E. coli whole cell after exposure to
formaldehyde (Figure S4). In both cases, the studies revealed a doseꢀdependent
formation of thioproline. As shown in Figure 4, cellꢀfree protein exposed to
formaldehyde produced 3.0 (±0.1) thioproline per 104 amino acids/for every mM
concentration of formaldehyde used. Given that the occurrence of cysteine in E. coli
protein was reported to be 1.1%,35 this result translated to 2.7 % of cysteine residues
modified per mM of formaldehyde exposure.
The amount of thioproline observed in protein isolated from E. coli treated with
formaldehyde was found to be 2.8 (±0.3) thioproline per 104 amino acids/mM
formaldehyde, which is comparable to the result of the cellꢀfree exposure.The
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excellent agreement of thioproline content in both cases indicated that the thioproline
formation in E.coli is mostly resulted from nonꢀenzymatic chemical reactions and
unaffected by the cellular environment.
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Quantitation of Proteinic Thioproline in Oxidant-Exposed E. coli Protein
Extract. The feasibility of detecting thiazolidination as a protein modification in
cellular protein was extended to quantitate thioproline in protein isolated from E. coli
cells exposed to hydroxyl radical generator of H2O2, Fe2+ꢀEDTA, and NaOCl to
simulate an oxidative environment for the cells. It is wellꢀknown that the hydroxyl
radical generators will produce hydroxyl radicals which will react with lipids and
proteins to generate formaldehyde.12, 36 The results of this analysis are shown in
Figure 5, which again showed a doseꢀdependent formation of thioproline in protein
samples isolated from the cells treated in the oxidative conditions. The
thiazolidination frequencies observed was 0.38 (±0.01) per 104 amino acids per µM
NaOCl, 0.6 (±0.05) per 104 amino acids per mM Fe2+ꢀEDTA, and 0.022 (±0.004) per
104 amino acids per µM H2O2 in proteins isolated from toxicantꢀexposed E. coli cells.
A similar relative reactivity in the order of NaOCl > H2O2 > Fe2+ꢀEDTA was also
observed in our previous study.23
Comparison with N6-formyllysine. The results of postꢀtranslational
thiazolidination to cysteine were compared with that of the wellꢀstudied lysine
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formylation process.14,37 To this end, we have also quantitated using LC–MS/MS, the
amount of N6ꢀformyllysine in formaldehydeꢀtreated protein (Figure 4) and in the
protein samples from the E. coli cells treated with RONS generators (Figure S5). The
study revealed the relative quantities of thioproline were 9ꢀ25% that of the
N6ꢀformyllysine levels (Table 1).
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This observed discrepancy in the levels of formylation of lysine and
thiazolidination of cysteine could have been attributed by to two main factors. Firstly,
the amino acid composition in the protein should be taken into consideration. It was
reported that the level of lysine is 2.2 times higher than that of cysteine in protein.38ꢀ40
In addition, cysteines are often tied up in disulfide bonds in proteins and thus not all
cysteine residues would be reactive.41,42 Therefore, it is expected that the chances of
N6ꢀformyllysine formation would be higher than for thioproline (Figure 1). Secondly, ,
as indicated in the studies using formaldehydeꢀtreated protein and E. coli (Table 1)
N6ꢀformylation of lysine is both chemically driven as well as and enzymatically
catalyzed, while there is currently no evidence that an enzyme would catalyse
enzymatic pathway for the thiazolidination reaction. Therefore, it is reasonable to
expect a larger amount of N6ꢀformyllysine than thioproline to be produced in these
experiments.
,
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The study revealed that the thiazolidination product, though detected to be
formed at a lower level than N6ꢀformyllysine, is nevertheless present in a significant
amount in both the in vitro and E. coli experiments. Despite the unknown biological
implications, it is feasible that cysteine thiazolidination could serve as a biomarker for
oxidative stress exposure to formaldehyde and it also has the potential of filling one of
the gaps in mining for unknown/novel protein modifications in proteins. However, it
is possible that thioproline modification may be present in proteins analyzed from
tissue fixed with formaldehyde. Thus, caution has to be taken if this biomarker was
chosen analyze in formaldehydeꢀfixed tissue samples.
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CONCLUSION
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We report in this study the initial identification of a novel protein modification,
cysteine thiazolidination, caused by formaldehyde. Using an isotope dilution LC–MS3
method of high sensitivity and selectivity, we quantitated the absolute and relative
quantity of thiazolidination in formaldehydeꢀtreated protein and in proteins isolated
from E. coli whole cells that were exposed to formaldehyde and oxidative stress
factors. The results from our study suggest the possibility of using cysteine
thiazolidination in proteins as a potential biomarker for oxidative stress and
formaldehyde exposure. It is believed that thioproline may be a better biomarker
candidate for oxidative stress exposure than N6ꢀformyllysine, because the latter can be
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confused with
N,Nꢀdimethyllysine, a rather common physiological PTM that has the
same nominal mass.
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Corresponding Author
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* Eꢀmail: chanwan@ust.hk
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Notes
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The authors declare no competing financial interest.
Acknowledgements
We thank Miss Yao Zhao (Summer research student from Kuang Yaming Honors
School, Nanjing University) for her assistance with the protein isolation and digestion.
We also thank AB Sciex for providing the LCꢀMS system for the study.
Funding Sources
This research was supported by the Research Grant Council of Hong Kong (grant
16313916). W. Chan thanks the Hong Kong University of Science and Technology
for Startup Funding (grant R9310).
Author Contributions
W. Chan and J. L. designed research; J. L. performed research; W. Chan, J. L., and K.
K. J. C. analyzed data; and W. Chan wrote the paper.
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Abbreviations
PTMs, Postꢀtranslational modifications; LC−MS3, liquid chromatographyꢀtandem
mass spectrometry; FmocꢀCl, fluorenylmethyloxycarbonyl chloride; MS,
Highꢀresolution mass spectrometry; MS/MS, product ion scan mass spectrometry;
RPꢀHPLC, reversedꢀphase high performance liquid chromatography; MRM,
multipleꢀreaction monitoring
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Supporting Information Available
Optimization of the molar ratios of FmocꢀCl over thioproline for the derivatization.
Method validation with customꢀsynthesized thioprolineꢀcontaining peptide.
Comparative analysis of thioproline in protein from formaldehydeꢀtreated E. coli by
LCꢀMS/MS and LCꢀMS3. LCꢀMS3 analyses of authentic Fmocꢀthioproline, authentic
thioproline after derivatizing with FmocꢀCl, thioproline in protein treated with
formaldehyde, and in protein in E. coli treated with formaldehyde. Doseꢀdependent
formation of N6ꢀformyllysine in protein in E. coli treated with Fe2+ꢀEDTA, NaOCl,
and H2O2. This information is available free of charge via the Internet at
http://pubs.acs.org.
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