Automated phosphopeptide enrichment from minute quantities of frozen malignant melanoma tissue
Murillo, Jimmy Rodriguez LU ; Kuras, Magdalena LU
; 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.
(Less)
- author
- Murillo, Jimmy Rodriguez
LU
; Kuras, Magdalena
LU
; Rezeli, Melinda
LU
; Milliotis, Tasso
LU
; Betancourt, Lazaro
LU
and Marko-Varga, Gyorgy
LU
- organization
- publishing date
- 2018-12-10
- 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
- 2024-04-15 20:15:02
@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}},
}