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Automated phosphopeptide enrichment from minute quantities of frozen malignant melanoma tissue

Murillo, Jimmy Rodriguez LU ; Kuras, Magdalena LU orcid ; Rezeli, Melinda LU ; Milliotis, Tasso LU ; Betancourt, Lazaro LU and Marko-Varga, Gyorgy LU (2018) In PLoS ONE 13(12).
Abstract

To acquire a deeper understanding of malignant melanoma (MM), it is essential to study the proteome of patient tissues. In particular, phosphoproteomics of MM has become of significant importance because of the central role that phosphorylation plays in the development of MM. Investigating clinical samples, however, is an extremely challenging task as there is usually only very limited quantities of material available to perform targeted enrichment approaches. Here, an automated phosphopeptide enrichment protocol using the AssayMap Bravo platform was applied to MM tissues and assessed for performance. The strategy proved to be highly-sensitive, less prone to variability, less laborious than existing techniques and adequate for starting... (More)

To acquire a deeper understanding of malignant melanoma (MM), it is essential to study the proteome of patient tissues. In particular, phosphoproteomics of MM has become of significant importance because of the central role that phosphorylation plays in the development of MM. Investigating clinical samples, however, is an extremely challenging task as there is usually only very limited quantities of material available to perform targeted enrichment approaches. Here, an automated phosphopeptide enrichment protocol using the AssayMap Bravo platform was applied to MM tissues and assessed for performance. The strategy proved to be highly-sensitive, less prone to variability, less laborious than existing techniques and adequate for starting quantities at the microgram level. An Fe(III)-NTA-IMAC-based enrichment workflow was applied to a dilution series of MM tissue lysates. The workflow was efficient in terms of sensitivity, reproducibility and phosphosite localization; and from only 12.5 μg of sample, more than 1,000 phosphopeptides were identified. In addition, from 60 μg of protein material the number of identified phosphoproteins from individual MM samples was comparable to previous reports that used extensive fractionation methods. Our data set included key pathways that are involved in MM progression; such as MAPK, melanocyte development and integrin signaling. Moreover, tissue-specific immunological proteins were identified, that have not been previously observed in the proteome of MM-derived cell lines. In conclusion, this workflow is suitable to study large cohorts of clinical samples that demand automatic and careful handling.

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publishing date
type
Contribution to journal
publication status
published
subject
in
PLoS ONE
volume
13
issue
12
article number
e0208562
publisher
Public Library of Science (PLoS)
external identifiers
  • scopus:85058239753
  • pmid:30532160
ISSN
1932-6203
DOI
10.1371/journal.pone.0208562
language
English
LU publication?
yes
id
62a65ba8-e675-481f-8e8f-e7dd98b17230
date added to LUP
2019-01-04 08:21:03
date last changed
2021-10-06 05:02:22
@article{62a65ba8-e675-481f-8e8f-e7dd98b17230,
  abstract     = {<p>To acquire a deeper understanding of malignant melanoma (MM), it is essential to study the proteome of patient tissues. In particular, phosphoproteomics of MM has become of significant importance because of the central role that phosphorylation plays in the development of MM. Investigating clinical samples, however, is an extremely challenging task as there is usually only very limited quantities of material available to perform targeted enrichment approaches. Here, an automated phosphopeptide enrichment protocol using the AssayMap Bravo platform was applied to MM tissues and assessed for performance. The strategy proved to be highly-sensitive, less prone to variability, less laborious than existing techniques and adequate for starting quantities at the microgram level. An Fe(III)-NTA-IMAC-based enrichment workflow was applied to a dilution series of MM tissue lysates. The workflow was efficient in terms of sensitivity, reproducibility and phosphosite localization; and from only 12.5 μg of sample, more than 1,000 phosphopeptides were identified. In addition, from 60 μg of protein material the number of identified phosphoproteins from individual MM samples was comparable to previous reports that used extensive fractionation methods. Our data set included key pathways that are involved in MM progression; such as MAPK, melanocyte development and integrin signaling. Moreover, tissue-specific immunological proteins were identified, that have not been previously observed in the proteome of MM-derived cell lines. In conclusion, this workflow is suitable to study large cohorts of clinical samples that demand automatic and careful handling.</p>},
  author       = {Murillo, Jimmy Rodriguez and Kuras, Magdalena and Rezeli, Melinda and Milliotis, Tasso and Betancourt, Lazaro and Marko-Varga, Gyorgy},
  issn         = {1932-6203},
  language     = {eng},
  month        = {12},
  number       = {12},
  publisher    = {Public Library of Science (PLoS)},
  series       = {PLoS ONE},
  title        = {Automated phosphopeptide enrichment from minute quantities of frozen malignant melanoma tissue},
  url          = {http://dx.doi.org/10.1371/journal.pone.0208562},
  doi          = {10.1371/journal.pone.0208562},
  volume       = {13},
  year         = {2018},
}