Analytical Chemistry Chemical Cytometry Quantitates Superoxide
Analytical Chemistry Chemical Cytometry Quantitates Superoxide
Analytical Chemistry Chemical Cytometry Quantitates Superoxide
You also want an ePaper? Increase the reach of your titles
YUMPU automatically turns print PDFs into web optimized ePapers that Google loves.
Scheme 3. Fragmentation Mechanism of Protonated 2 upon CID, Delivering Two Complementary Doublets of 26 u<br />
Mass Shifted Product Ions a<br />
a Product ions of peptide 1 are 6a and 7a, and product ions of peptide 2 are 6b and 7b.<br />
Scheme 4. Fragmentation Mechanism of a Protonated Type 1 Modified Peptide (3) upon CID, Delivering a Product<br />
Ion That Is Modified with BuUr [M + H + BuUr] + (7) by a CNL of Pyrolidinone (85 u)<br />
Table 1. Summary of the Characteristic CNLs and Fragment Ion Mass Shifts Used for Discrimination of the<br />
Different Cross-Linking Types (ESI-Ion Trap-MS/MS and MALDI-TOF/TOF-MS/MS)<br />
cross-link type hydrolyzed, type 0 aminolyzed, type 0 intrapeptide, type 1 interpeptide, type 2<br />
CNL 103 and 129 u 102 and 128 u 85 u<br />
no. of 26 u doublets 1 1 2<br />
the latter presents the respective product ion spectrum of [M<br />
+ H + BuUr] + at m/z 969.53 (7). In both experiments the<br />
product ion analysis was conducted in the orbitrap at a mass<br />
accuracy of 1-2 ppm employing a resolving power of 15 000.<br />
In the MS 3 product ion spectra presented in Figure 1B,C, the<br />
precursor ions exhibit frequent losses of water and ammonia, and<br />
more importantly, extensive backbone cleavages allow an efficient<br />
sequencing of the peptide. In the present case, b-type ions<br />
containing the cross-linker dominate the spectra. From the CID<br />
fragmentation pattern (Figure 1A) it is immediately obvious that<br />
fragmentation of the urea cross-linker is strongly preferred, while<br />
the peptide backbone remains virtually intact. The effective<br />
fragmentation of the partially hydrolyzed cross-linker 4 leads to<br />
the formation of prominent “26 u doublet” product ions 6 and 7<br />
(Table 1). As highlighted above, these product ions are indicative<br />
of a selective and sensitive detection of the respective cross-linked<br />
6962 <strong>Analytical</strong> <strong>Chemistry</strong>, Vol. 82, No. 16, August 15, 2010<br />
species as they cannot be confused with b- and corresponding<br />
a-type ions giving a mass difference of 28 u (CO). Peptides that<br />
are modified by an amidated cross-linker (5), resulting from the<br />
quenching reaction with ammonium bicarbonate, are also observed<br />
in the product ion mass spectra, 37 accordingly exhibiting<br />
CNLs of 128 and 102 u (Scheme 2, Table 1). It should be noted<br />
that peptides modified by hydrolyzed (4) and amidated (5) linkers<br />
were readily separated by our nano-HPLC method with ca. 30 s<br />
elution time difference for a 1000 Da peptide.<br />
An analogous fragmentation behavior was observed for a<br />
modified tryptic peptide of PPARR comprising amino acids<br />
259-272. ESI-CID-MS 2 experiments resulted in characteristic<br />
CNLs of 129 and 103 u with the indicative 26 u pattern due to<br />
(37) Kalkhof, S.; Sinz, A. Anal. Bioanal. Chem. 2008, 392, 305–312.