Direct Identification of Tyrosine Sulfation by using Ultraviolet Photodissociation Mass Spectrometry
Michelle R. Robinson, Kevin L. Moore, and Jennifer S. Brodbelt
Journal of The American Society for Mass Spectrometry
Supplemental Figures
Figure and Table Legends:
Supplemental Figure 1. Electrospray ionization efficiency of sulfated cholecystokinin, RDsYTGWNleDFNH2 in positive ion mode. Cholecystokinin was prepared at a concentration of 10 uM in 25% methanol
with the following solvent additives: 0.1% formic acid (A), 5mM ammonium acetate (B). Each spectrum
is the average of 20 FTMS scans. Cholecystokinin ions, M+H and M+2H, are annotated in green while
various salt adducts of these ions are annotated in blue. Ions that exhibit neutral loss of SO3 are
annotated in red.
Supplemental Figure 2. MS/MS fragmentation behavior of singly protonated cholecystokinin,
RDsYTGWLDF-NH2 (1+). CID (A) and HCD (B), each at 35% normalized collision energy, result in
predominant neutral loss of SO3. HCD promotes more backbone fragmentation compared to CID
accounting for 100% sequence coverage; however, all product ions lack the sulfate modification. UVPD
(C) using three 2 mJ laser pulses generates an extensive array of a and x fragment ions accounting for
75% sequence coverage, although SO3 is lost from each ion. Additionally, neutral loss of the entire
sulfotyrosine side chain (CH2C6H4HSO4) was observed upon UVPD and annotated as “-sY”. In all spectra,
neutral loss of SO3 or sY are annotated in red.
Supplemental Figure 3. (A) Sequence ions produced using 193 nm UVPD. (B) Homolytic bond cleavage at
C-Cα bond to produce a+1 and x+1 radical ions. (C) Radical ions may undergo subsequent loss of radical
protons to form even electron a and x type product ions. All product ions are generated upon UVPD of
peptide cations. For UVPD of peptide anions, a and x ions (both radical and even electron species) are
more commonly observed compared to other backbone cleavages.
Supplemental Figure 4. FT isolation of 2+ sulfated cholecystokinin for MS/MS analysis. The full mass
spectrum from m/z 580-660 is displayed in the top panel with 2+ cholecystokinin annotated at m/z 626.
Various isolation widths were applied in each subsequent spectrum without the addition of normalized
collision energy. A dramatic decrease in the abundance of 2+ cholecystokinin is observed using the
smallest isolation width of 2, which impedes subsequent MS/MS activation. Additionally, the ion
isolation process promotes sulfate loss to a small degree further demonstrating the lability of the
sulfoester bond.
Supplemental Figure 5. MS/MS fragmentation behavior of doubly protonated cholecystokinin,
RDsYTGWLDF-NH2 (2+). Upon CID at 35% normalized collision energy (NCE) (A), the primary product was
neutral loss of SO3. For HCD at 35% NCE (B), an array of b and a ions were generated, though all lacked
the sulfate modification. UVPD using three 2 mJ laser pulses (C) generates an extensive array of a and x
fragment ions accounting for 75% sequence coverage, although SO3 is lost from each ion. Neutral losses
of SO3 are annotated in red. ETD using a 100 ms activation time (D) resulted in backbone fragmentation
without loss of modification; however, limited sequence coverage was achieved.
Supplemental Figure 6. Electrospray ionization efficiency of sulfated cholecystokinin, RDsYTGWNleDFNH2 in negative ion mode. Cholecystokinin was prepared at a concentration of 10 uM in 25% methanol
with the following solvent additives: 5 mM ammonium acetate (A), 0.1% ammonium hydroxide (B). Each
spectrum is the average of 20 FTMS scans. Cholecystokinin M+2H ions are annotated in green. Sodium
and potassium adducts to the precursor ion are annotated in blue.
Supplemental Figure 7. HCD fragmentation of doubly deprotonated cholecystokinin, RDsYTGWLDFNH2(2-), m/z 624.24 using NCE 35% (top) and NCE 55% (bottom). Neutral loss of H2O and CO2 from the
precursor ion are the most abundant products at 35% NCE. At 55% NCE, two products, a7 and c7, derived
from backbone cleavage N-terminal to aspartic acid are also observed. All products that lose the
sulfated modification are annotated in red.
Supplemental figure 8. UVPD of doubly sulfated cionin in1- (A), 2- (B), and 3- (C) charge states. Loss of
one SO3 modification is observed from all product ions. The a2 ion, which is expected to only contain
one SO3, is the only product ion that does not experience neutral loss of sulfate. The product ion labeled
“-sY” denotes a loss of the entire sulfotyrosine side chain from the charge reduced precursor ion,
corresponding to loss of CH2C6H4HSO4
Supplemental figure 9. Charge state distribution of sulfopeptides caerulein, cholecystokinin, cionin,
hirudin, and leu-enkephalin in (A) basic (0.1% ammonium hydroxide) and (B) neutral (5mM ammonium
acetate) solutions.
Supplemental figure 10. Laser parameter optimization evaluated based on the degree of SO3 loss from
the precursor or charge reduced precursor ions. The abundances of neutral loss ions were tracked
across all laser conditions for each peptide and normalized to 100%, which is represented as the color
red in the heat maps (0% is blue in color). Heat maps for 1- and 2- precursor ions (top and middle,
respectively) contain data from all four peptides, whereas heat maps for 3- precursor ions (bottom)
contain data only from hirudin and caerulein. The deepest red shade indicates the most optimum
outcome.
Supplemental figure 11. MS1 LOD determined for leu-enkephalin (1-), caerulein (2-), and cionin (2-) A)
Extracted ion chromatograms (XIC) for injections of 1500, 1250, 1000, 750, 500, and 250 fmol of
peptide. B) The area under the curve was plotted as a function of femtomoles of peptide injected to
generate a calibration curve for each peptide. C) MS1 LOD was determined for each peptide using the
calibration curve slope and the standard deviation of the average peak area.
Supplemental figure 12. UVPD for (A) leu-enkephalin, (B) caerulein, and (C) cionin in 100 and 250
femtomole injections of the equimolar peptide mixture. In each set of spectra, the three a ions used for
MS2 LOD are highlighted in green, the precursor and charge reduced precursor and highlighted in blue,
and the neutral loss of SO3 from the precursor is highlighted in red. Leu-enkephalin sequence ions are
detected at both 250 and 100 fmol, while caerulein and cionin sequence ions are not detected below
250 fmol. The neutral loss of SO3 is abundant in each spectrum making it a useful marker for sulfation.
Supplemental Figure 1. Electrospray ionization efficiency of sulfated cholecystokinin, RDsYTGWNleDF-NH2 in
positive ion mode. Cholecystokinin was prepared at a concentration of 10 uM in 25% methanol with the following
solvent additives: (A) 0.1% formic acid, (B) 5 mM ammonium acetate. Each spectrum is the average of 20 FTMS
scans. Cholecystokinin ions, M+H and M+2H, are annotated in green while various salt adducts of these ions are
annotated in blue. Ions that exhibit neutral loss of SO3 are annotated in red.
Supplemental Figure 2. MS/MS fragmentation behavior of singly protonated cholecystokinin, RDsYTGWLDF-NH2
(1+). CID (A) and HCD (B), each at 35% normalized collision energy, result in predominant neutral loss of SO3. HCD
promotes more backbone fragmentation compared to CID accounting for 100% sequence coverage; however, all
product ions lack the sulfate modification. UVPD (C) using three 2 mJ laser pulses generates an extensive array of a
and x fragment ions accounting for 75% sequence coverage, although SO3 is lost from each ion. Additionally, neutral
loss of the entire sulfotyrosine side chain (CH2C6H4HSO4) was observed upon UVPD and annotated as “-sY”. In all
spectra, neutral loss of SO3 or sY are annotated in red.
Supplemental Figure 3. (A) Sequence ions produced using 193 nm UVPD. (B) Homolytic bond cleavage at C-Cα
bond to produce a+1 and x+1 radical ions. (C) Radical ions may undergo subsequent loss of radical protons to form
even electron a and x type product ions. All product ions are generated upon UVPD of peptide cations. For UVPD of
peptide anions, a and x ions (both radical and even electron species) are more commonly observed compared to
other backbone cleavages.
Supplemental Figure 4. FT isolation of 2+ sulfated cholecystokinin for MS/MS analysis. The ESI mass spectrum
from m/z 580-660 is displayed in the top panel with 2+ cholecystokinin annotated at m/z 626. Various isolation widths
were applied in each subsequent spectrum without the addition of normalized collision energy. A dramatic decrease
in the abundance of 2+ cholecystokinin is observed using the smallest isolation width of 2, which impedes
subsequent MS/MS activation. Additionally, the ion isolation process promotes sulfate loss to a small degree further
demonstrating the lability of the sulfoester bond.
Supplemental Figure 5. MS/MS fragmentation behavior of doubly protonated cholecystokinin, RDsYTGWLDF-NH2
(2+). Upon CID at 35% normalized collision energy (NCE) (A), the primary product was neutral loss of SO3. For HCD
at 35% NCE (B), an array of b and a ions were generated, though all lacked the sulfate modification. UVPD using
three 2 mJ laser pulses (C) generates an extensive array of a and x fragment ions accounting for 75% sequence
coverage, although SO3 is lost from each ion. Neutral losses of SO3 are annotated in red. ETD using a 100 ms
activation time (D) resulted in backbone fragmentation without loss of modification; however, limited sequence
coverage was achieved.
Supplemental Figure 6. Electrospray ionization efficiency of sulfated cholecystokinin, RDsYTGWNleDF-NH2 in
negative ion mode. Cholecystokinin was prepared at a concentration of 10 uM in 25% methanol with the following
solvent additives: 5 mM ammonium acetate (A), 0.1% ammonium hydroxide (B). Each spectrum is the average of 20
FTMS scans. Cholecystokinin M+2H ions are annotated in green. Sodium and potassium adducts to the precursor
ion are annotated in blue.
Supplemental Figure 7. HCD fragmentation of doubly deprotonated cholecystokinin, RDsYTGWLDF-NH2(2-), m/z
624.24 using NCE 35% (top) and NCE 55% (bottom). Neutral loss of H2O and CO2 from the precursor ion are the
most abundant products at 35% NCE. At 55% NCE, two products, a7 and c7, derived from backbone cleavage Nterminal to aspartic acid are also observed. All products that lose the sulfated modification are annotated in red.
Supplemental figure 8. UVPD of doubly sulfated cionin in (A) 1-, (B) 2-, and (C) 3- charge states. Loss of one SO3
modification is observed from all product ions. The a2 ion, which is expected to only contain one SO3, is the only
product ion that does not experience neutral loss of sulfate. The product ion labeled “-sY” denotes a loss of the entire
sulfotyrosine side chain from the charge reduced precursor ion, corresponding to loss of CH2C6H4HSO4
Supplemental figure 9. Charge state distribution of sulfopeptides caerulein, cholecystokinin, cionin, hirudin, and leuenkephalin in (A) basic (0.1% ammonium hydroxide) and (B) neutral (5mM ammonium acetate) solutions.
Supplemental figure 10. Laser parameter optimization evaluated based on the degree of SO3 loss from the
precursor or charge reduced precursor ions. The abundances of neutral loss ions were tracked across all laser
conditions for each peptide and normalized to 100%, which is represented as the color red in the heat maps (0% is
blue in color). Heat maps for 1- and 2- precursor ions (top and middle, respectively) contain data from all four
peptides, whereas heat maps for 3- precursor ions (bottom) contain data only from hirudin and caerulein. The
deepest red shade indicates the most optimum outcome.
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Supplemental figure 11. MS LOD determined for leu-enkephalin (1-), caerulein (2-), and cionin (2-) A) Extracted
ion chromatograms (XIC) for injections of 1500, 1250, 1000, 750, 500, and 250 fmol of peptide. B) The area under
the curve was plotted as a function of femtomoles of peptide injected to generate a calibration curve for each peptide.
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C) MS LOD was determined for each peptide using the calibration curve slope and the standard deviation of the
average peak area.
Supplemental figure 12. UVPD for (A) leu-enkephalin, (B) caerulein, and (C) cionin in 100 and 250 femtomole
injections of the equimolar peptide mixture. In each set of spectra, the three a ions used for MS2 LOD are highlighted
in green, the precursor and charge reduced precursor and highlighted in blue, and the neutral loss of SO3 from the
precursor is highlighted in red. Leu-enkephalin sequence ions are detected at both 250 and 100 fmol, while caerulein
and cionin sequence ions are not detected below 250 fmol. The neutral loss of SO3 is abundant in each spectrum
making it a useful marker for sulfation.