8. It is critical to wash away the remaining 50 mM EDTA for
phosphopeptide binding to IMAC tip.
9. The IMAC loading buffer must be freshly prepared because of
high concentration of ACN. The ACN concentration will
affect the specificity of phosphopeptide enrichment.
10. The IMAC wash buffer must be freshly prepared because of
high concentration of ACN. The ACN concentration will
affect the specificity of phosphopeptide enrichment.
11. It is critical to wash away the remaining ACN in the IMAC tip
because it will affect the recovery of phosphopeptides for C18
desalting.
12. For C18 StageTip, the membrane disk is soft and seals very
easily at its sides. Applying a larger pressure at this point results
in a more densely packed disk and raises the back pressure of
the StageTip unnecessarily.
Acknowledgments
Portions of the research were supported by P41GM103493 (R.D.
S), R21CA223715 (T.S.), T32GM7171 (J.S.S.), the Duke Medical
Scientist Training Program (J.S.S.), R01GM122798 (S.R.), and
Burroughs Wellcome Career Award for Medical Scientists (S.R.).
The experimental work described herein was performed in the
Environmental Molecular Sciences Laboratory, Pacific Northwest
National Laboratory, a national scientific user facility sponsored by
the United States of America Department of Energy under Contract DE-AC05-76RL0 1830.
References
1. Graves JD, Krebs EG (1999) Protein phosphorylation and signal transduction. Pharmacol Ther 82:111–121
2. Day EK, Sosale NG, Lazzara MJ (2016) Cell
signaling regulation by protein phosphorylation: a multivariate, heterogeneous, and
context-dependent process. Curr Opin Biotech
40:185–192
3. Dermit M, Dokal A, Cutillas PR (2017)
Approaches to identify kinase dependencies in
cancer signalling networks. FEBS Lett
591:2577–2592
4. Ardito F, Giuliani M, Perrone D, Troiano G,
Lo Muzio L (2017) The crucial role of protein
phosphorylation in cell signaling and its use as
targeted therapy. Int J Mol Med 40:271–280
5. Riley NM, Coon JJ (2016) Phosphoproteomics in the age of rapid and deep proteome
profiling. Anal Chem 88:74–94
6. Tsai CF, Wang YT, Yen HY, Tsou CC, Ku WC,
Lin PY, Chen HY, Nesvizhskii AI, Ishihama Y,
Chen YJ (2015) Large-scale determination of
absolute phosphorylation stoichiometries in
human cells by motif-targeting quantitative
proteomics. Nat Commun 6:6622
7. Humphrey SJ, Azimifar SB, Mann M (2015)
High-throughput phosphoproteomics reveals
in vivo insulin signaling dynamics. Nat Biotechnol 33:990–995
8. Bekker-Jensen DB, Kelstrup CD, Batth TS,
Larsen SC, Haldrup C, Bramsen JB, Sorensen
KD, Hoyer S, Orntoft TF, Andersen CL, Nielsen ML, Olsen JV (2017) An optimized shotgun strategy for the rapid generation of
comprehensive human proteomes. Cell Syst
4:587–599 e584
9. Wakabayashi M, Kyono Y, Sugiyama N, Ishihama Y (2015) Extended coverage of singly
256
Chia-Feng Tsai et al.
phosphopeptide binding to IMAC tip.
9. The IMAC loading buffer must be freshly prepared because of
high concentration of ACN. The ACN concentration will
affect the specificity of phosphopeptide enrichment.
10. The IMAC wash buffer must be freshly prepared because of
high concentration of ACN. The ACN concentration will
affect the specificity of phosphopeptide enrichment.
11. It is critical to wash away the remaining ACN in the IMAC tip
because it will affect the recovery of phosphopeptides for C18
desalting.
12. For C18 StageTip, the membrane disk is soft and seals very
easily at its sides. Applying a larger pressure at this point results
in a more densely packed disk and raises the back pressure of
the StageTip unnecessarily.
Acknowledgments
Portions of the research were supported by P41GM103493 (R.D.
S), R21CA223715 (T.S.), T32GM7171 (J.S.S.), the Duke Medical
Scientist Training Program (J.S.S.), R01GM122798 (S.R.), and
Burroughs Wellcome Career Award for Medical Scientists (S.R.).
The experimental work described herein was performed in the
Environmental Molecular Sciences Laboratory, Pacific Northwest
National Laboratory, a national scientific user facility sponsored by
the United States of America Department of Energy under Contract DE-AC05-76RL0 1830.
References
1. Graves JD, Krebs EG (1999) Protein phosphorylation and signal transduction. Pharmacol Ther 82:111–121
2. Day EK, Sosale NG, Lazzara MJ (2016) Cell
signaling regulation by protein phosphorylation: a multivariate, heterogeneous, and
context-dependent process. Curr Opin Biotech
40:185–192
3. Dermit M, Dokal A, Cutillas PR (2017)
Approaches to identify kinase dependencies in
cancer signalling networks. FEBS Lett
591:2577–2592
4. Ardito F, Giuliani M, Perrone D, Troiano G,
Lo Muzio L (2017) The crucial role of protein
phosphorylation in cell signaling and its use as
targeted therapy. Int J Mol Med 40:271–280
5. Riley NM, Coon JJ (2016) Phosphoproteomics in the age of rapid and deep proteome
profiling. Anal Chem 88:74–94
6. Tsai CF, Wang YT, Yen HY, Tsou CC, Ku WC,
Lin PY, Chen HY, Nesvizhskii AI, Ishihama Y,
Chen YJ (2015) Large-scale determination of
absolute phosphorylation stoichiometries in
human cells by motif-targeting quantitative
proteomics. Nat Commun 6:6622
7. Humphrey SJ, Azimifar SB, Mann M (2015)
High-throughput phosphoproteomics reveals
in vivo insulin signaling dynamics. Nat Biotechnol 33:990–995
8. Bekker-Jensen DB, Kelstrup CD, Batth TS,
Larsen SC, Haldrup C, Bramsen JB, Sorensen
KD, Hoyer S, Orntoft TF, Andersen CL, Nielsen ML, Olsen JV (2017) An optimized shotgun strategy for the rapid generation of
comprehensive human proteomes. Cell Syst
4:587–599 e584
9. Wakabayashi M, Kyono Y, Sugiyama N, Ishihama Y (2015) Extended coverage of singly
256
Chia-Feng Tsai et al.