In-gel and in-solution approaches to protein and peptide fractionation

Dylan H. Multari; Mohadeseh Montazeri Shatouri; Sabrina Grizzi de Oliveira; Zeshan Ali; Fatemeh Habibpourmehraban; Farhad Masoomi-Aladizgeh; Paul A. Haynes

doi:10.1007/978-1-0716-4298-6_3

In-gel and in-solution approaches to protein and peptide fractionation

Dylan H. Multari, Mohadeseh Montazeri Shatouri, Sabrina Grizzi de Oliveira, Zeshan Ali, Fatemeh Habibpourmehraban, Farhad Masoomi-Aladizgeh, Paul A. Haynes^*

^*Corresponding author for this work

School of Natural Sciences

Research output: Chapter in Book/Report/Conference proceeding › Chapter › peer-review

1 Citation (Scopus)

Abstract

Identifying proteins from living organisms helps us understand the biological functions of cells, discover new molecular mechanisms, and interrogate known mechanisms for improving our understanding. For a comprehensive understanding of cellular functions, identifying the whole protein content, or proteome, of a cell is desirable but challenging. Here, we describe in detail two methods of proteome fractionation at either the protein (SDS-PAGE) or peptide (high-pH reversed-phase fractionation) level, which can be used to maximize the identification of proteins from complex biological samples. We apply these methods to two different sample types commonly processed in our laboratory to demonstrate the versatility of these protocols. These methods produce many more peptide identifications when compared to conventional single-shot analysis and can also be used in combination to generate larger complementary datasets with greater depth of proteome coverage.

Original language	English
Title of host publication	Methods in Molecular Biology
Editors	Taufika Islam Williams
Place of Publication	New York, NY
Publisher	Springer, Springer Nature
Chapter	3
Pages	25-41
Number of pages	17
ISBN (Electronic)	9781071642986
ISBN (Print)	9781071642979
DOIs	https://doi.org/10.1007/978-1-0716-4298-6_3
Publication status	Published - 2025

Publication series

Name	Methods in Molecular Biology
Volume	2884
ISSN (Print)	1064-3745
ISSN (Electronic)	1940-6029

Keywords

Filter-aided sample preparation (FASP)
High-pH reversed-phase fractionation
In-gel digestion
Proteomics
Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

Access to Document

10.1007/978-1-0716-4298-6_3

Cite this

Multari, D. H., Shatouri, M. M., de Oliveira, S. G., Ali, Z., Habibpourmehraban, F., Masoomi-Aladizgeh, F., & Haynes, P. A. (2025). In-gel and in-solution approaches to protein and peptide fractionation. In T. I. Williams (Ed.), Methods in Molecular Biology (pp. 25-41). (Methods in Molecular Biology; Vol. 2884). Springer, Springer Nature. https://doi.org/10.1007/978-1-0716-4298-6_3

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abstract = "Identifying proteins from living organisms helps us understand the biological functions of cells, discover new molecular mechanisms, and interrogate known mechanisms for improving our understanding. For a comprehensive understanding of cellular functions, identifying the whole protein content, or proteome, of a cell is desirable but challenging. Here, we describe in detail two methods of proteome fractionation at either the protein (SDS-PAGE) or peptide (high-pH reversed-phase fractionation) level, which can be used to maximize the identification of proteins from complex biological samples. We apply these methods to two different sample types commonly processed in our laboratory to demonstrate the versatility of these protocols. These methods produce many more peptide identifications when compared to conventional single-shot analysis and can also be used in combination to generate larger complementary datasets with greater depth of proteome coverage.",

keywords = "Filter-aided sample preparation (FASP), High-pH reversed-phase fractionation, In-gel digestion, Proteomics, Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE)",

author = "Multari, {Dylan H.} and Shatouri, {Mohadeseh Montazeri} and {de Oliveira}, {Sabrina Grizzi} and Zeshan Ali and Fatemeh Habibpourmehraban and Farhad Masoomi-Aladizgeh and Haynes, {Paul A.}",

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Multari, DH, Shatouri, MM , de Oliveira, SG , Ali, Z, Habibpourmehraban, F, Masoomi-Aladizgeh, F & Haynes, PA 2025, In-gel and in-solution approaches to protein and peptide fractionation. in TI Williams (ed.), Methods in Molecular Biology. Methods in Molecular Biology, vol. 2884, Springer, Springer Nature, New York, NY, pp. 25-41. https://doi.org/10.1007/978-1-0716-4298-6_3

In-gel and in-solution approaches to protein and peptide fractionation. / Multari, Dylan H.; Shatouri, Mohadeseh Montazeri ; de Oliveira, Sabrina Grizzi et al.
Methods in Molecular Biology. ed. / Taufika Islam Williams. New York, NY: Springer, Springer Nature, 2025. p. 25-41 (Methods in Molecular Biology; Vol. 2884).

Research output: Chapter in Book/Report/Conference proceeding › Chapter › peer-review

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AU - Multari, Dylan H.

AU - Shatouri, Mohadeseh Montazeri

AU - de Oliveira, Sabrina Grizzi

AU - Ali, Zeshan

AU - Habibpourmehraban, Fatemeh

AU - Masoomi-Aladizgeh, Farhad

AU - Haynes, Paul A.

PY - 2025

Y1 - 2025

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AB - Identifying proteins from living organisms helps us understand the biological functions of cells, discover new molecular mechanisms, and interrogate known mechanisms for improving our understanding. For a comprehensive understanding of cellular functions, identifying the whole protein content, or proteome, of a cell is desirable but challenging. Here, we describe in detail two methods of proteome fractionation at either the protein (SDS-PAGE) or peptide (high-pH reversed-phase fractionation) level, which can be used to maximize the identification of proteins from complex biological samples. We apply these methods to two different sample types commonly processed in our laboratory to demonstrate the versatility of these protocols. These methods produce many more peptide identifications when compared to conventional single-shot analysis and can also be used in combination to generate larger complementary datasets with greater depth of proteome coverage.

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KW - High-pH reversed-phase fractionation

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KW - Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

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ER -

In-gel and in-solution approaches to protein and peptide fractionation

Abstract

Publication series

Keywords

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FAAB: ARC Training Centre for Facilitated Advancement of Australia's Bioactives (FAAB)

Cite this

In-gel and in-solution approaches to protein and peptide fractionation

Abstract

Publication series

Keywords

Access to Document

Other files and links

Projects

FAAB: ARC Training Centre for Facilitated Advancement of Australia's Bioactives (FAAB)

Cite this