Supporting information for
Investigating quantitation of phosphorylation using MALDI-TOF mass
spectrometry
Laurie Parker,* Aaron Engel-Hall, Kevin Drew, George Steinhardt,
Donald L. Helseth Jr., David Jabon, Timothy McMurry, David S. Angulo
and Stephen J. Kron
Table of contents:
1. Characterization data for peptide solutions
2. Additional representative mass spectra
1. Characterization data for peptide solutions
Peptides were analyzed on an Agilent ion trap LC-MS and peak areas for 214 nm
UV absorbance were determined using Agilent Chemstation software. Each peptide
was dissolved in 0.1% TFA to approximately 1 mM, assuming a starting amount of 1 mg
(based on supplier specifications). The “1 mM” stock solutions were diluted to 100 M,
and equal volumes of each unphosphorylated and phosphorylated peptide were injected
for analysis by LC-MS. The relative peak areas in the 215 nm UV absorbance
chromatograms were quantified, and a correction factor was calculated for the deviation
from 1:1 of their relative concentrations. This correction factor was applied to the
assumed “actual phosphopeptide input” values before plotting the calibration curves
(see spreadsheet for calculations). It should be noted that only the 215 nm absorbance
ratios of the peaks of interest were considered when calculating correction values, thus
purity was not a confounding factor in the “actual” input amounts.
HPLC separation conditions and injection volumes are given for each peptide
pair. Mass spectra shown are average mass spectra across the entire UV peak of
interest, except where otherwise noted. In the case of peptides 3 and 6, the
phosphorylated form contained more than one amide bond isomer at a significant
population, most likely the cis amide. Conformational restriction of peptides upon
phosphorylation, and consequent changes in isomeric populations, is known to result in
different retention times for the isomeric species in reverse-phase HPLC.1 In the work
described herein, the identities of isomers were validated using extracted ion
chromatograms to show that the additional peaks had the same mass as the expected
peptide, and were not from impurities in the sample.
1
LC-MS Spectra
1. PKTPKKAKKL (96% purity)
2
1-P. PKpTPKKAKKL (92% purity)
3
3. ADAQHATPPKKKRKVEDPKDF (98% purity)
4
3-P. ADAQHApTPPKKKRKVEDPKDF (93% purity)
5
3. MHRQETVACLK (75% purity)
6
3-P. MHRQEpTVACLK (90% purity): Extracted ion chromatogram for m/z 1439 is
shown to illustrate that both peaks visible in the UV are isomeric species of
MHRQEpTVACLK.
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4. RRLIEDNEYTARG (96% purity)
8
4-P. RRLIEDNEpYTARG (90% purity)
9
5. acFHDDSDEDLLHI (90% purity)
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5-P. acFHDDpSDEDLLHI (91% purity)
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6. TSTEPQYQPGENL (95% purity)
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6-P. TSTEPQpYQPGENL (95% purity). Extracted ion chromatogram detail to show that
the peptide TSTEPQpYQPGENL represents the bulk of the “double” peak seen with
215 nm UV detection. Double peak represents structural isomers of
TSTEPQpYQPGENL.
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7. ADEYLIPQQ (95% purity)
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7-P. ADEpYLIPQQ (90% purity)
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2. Additional representative mass spectra
Full spectra of representative examples for each peptide at ~35% phosphopeptide
inputs in DHB matrix.
16
Representative negative mode spectra for delayed extraction experiments using peptide
6 spotted with CHCA matrix.
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Representative full spectra comparing matrices for peptide 4 at 60.7% input
phosphopeptide in linear positive and negative modes.
1.
Guo, Y. T.; Li, Y. M.; Zhu, Z. T.; Zhao, Y. F., Effect of the phosphate group with
different negative charges on the conformation of phosphorylated Ser/Thr-Pro motif.
International Journal Of Peptide Research And Therapeutics 2005, 11, (2), 159-165.
18