Phosphotyrosine biased enrichment of tryptic peptides from cancer cells by combining pY-MIP and TiO2 affinity resins

Loreta Bllaci, Silje Bøen Torsetnes, Celina Katarzyna Wierzbicka, Sudhirkumar Shinde, Börje Sellergren, Adelina Rogowska-Wrzesinska, Ole Nørregaard Jensen

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Resumé

Protein phosphorylation at distinct tyrosine residues (pY) is essential for fast, specific and accurate signal transduction in cells. Enrichment of pY-containing peptides derived from phosphoproteins is commonly facilitated by use of immobilized anti-pY antibodies prior to phosphoproteomics analysis by mass spectrometry. We here report on an alternative approach for pY-peptide enrichment using inexpensive pY imprinted polymer (pY-MIP). We assessed by mass spectrometry the performance of pY-MIP for enrichment and sequencing of phosphopeptides obtained by tryptic digestion of protein extracts from HeLa cells. The combination of pY-MIP and TiO2 based phosphopeptide enrichment provided more than 90% selectivity for phosphopeptides. Mass spectrometry signal intensities were enhanced for most pY-phosphopeptides (approximately 70%) when using the pY-MIP-TiO2 combination as compared to TiO2 alone. pY constituted up to 8% of the pY-MIP-TiO2 enriched phosphopeptide fractions. The pY-MIP-TiO2 and the TiO2 protocols yielded comparable numbers of distinct phosphopeptides, 1693 and 1842 respectively, from microgram levels of peptide samples. Detailed analysis of physicochemical properties of pY-MIP-TiO2 enriched phosphopeptides demonstrated that this protocol retrieved phosphopeptides that tend to be smaller (<24 residues), less acidic and almost exclusively monophosphorylated, as compared to TiO2 alone. These unique properties render the pY-MIP based phosphopeptide enrichment technique an attractive alternative for applications in phosphoproteomics research.

OriginalsprogEngelsk
TidsskriftAnalytical Chemistry
Vol/bind89
Udgave nummer21
Sider (fra-til)11332–11340
ISSN0003-2700
DOI
StatusUdgivet - 2017

Fingeraftryk

Phosphopeptides
Phosphotyrosine
Polymers
Resins
Cells
Peptides
Mass spectrometry
Signal transduction
Phosphorylation
Phosphoproteins
Tyrosine
Proteins
Antibodies

Citer dette

Bllaci, Loreta ; Torsetnes, Silje Bøen ; Wierzbicka, Celina Katarzyna ; Shinde, Sudhirkumar ; Sellergren, Börje ; Rogowska-Wrzesinska, Adelina ; Jensen, Ole Nørregaard. / Phosphotyrosine biased enrichment of tryptic peptides from cancer cells by combining pY-MIP and TiO2 affinity resins. I: Analytical Chemistry. 2017 ; Bind 89, Nr. 21. s. 11332–11340.
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title = "Phosphotyrosine biased enrichment of tryptic peptides from cancer cells by combining pY-MIP and TiO2 affinity resins",
abstract = "Protein phosphorylation at distinct tyrosine residues (pY) is essential for fast, specific and accurate signal transduction in cells. Enrichment of pY-containing peptides derived from phosphoproteins is commonly facilitated by use of immobilized anti-pY antibodies prior to phosphoproteomics analysis by mass spectrometry. We here report on an alternative approach for pY-peptide enrichment using inexpensive pY imprinted polymer (pY-MIP). We assessed by mass spectrometry the performance of pY-MIP for enrichment and sequencing of phosphopeptides obtained by tryptic digestion of protein extracts from HeLa cells. The combination of pY-MIP and TiO2 based phosphopeptide enrichment provided more than 90{\%} selectivity for phosphopeptides. Mass spectrometry signal intensities were enhanced for most pY-phosphopeptides (approximately 70{\%}) when using the pY-MIP-TiO2 combination as compared to TiO2 alone. pY constituted up to 8{\%} of the pY-MIP-TiO2 enriched phosphopeptide fractions. The pY-MIP-TiO2 and the TiO2 protocols yielded comparable numbers of distinct phosphopeptides, 1693 and 1842 respectively, from microgram levels of peptide samples. Detailed analysis of physicochemical properties of pY-MIP-TiO2 enriched phosphopeptides demonstrated that this protocol retrieved phosphopeptides that tend to be smaller (<24 residues), less acidic and almost exclusively monophosphorylated, as compared to TiO2 alone. These unique properties render the pY-MIP based phosphopeptide enrichment technique an attractive alternative for applications in phosphoproteomics research.",
author = "Loreta Bllaci and Torsetnes, {Silje B{\o}en} and Wierzbicka, {Celina Katarzyna} and Sudhirkumar Shinde and B{\"o}rje Sellergren and Adelina Rogowska-Wrzesinska and Jensen, {Ole N{\o}rregaard}",
year = "2017",
doi = "10.1021/acs.analchem.7b02091",
language = "English",
volume = "89",
pages = "11332–11340",
journal = "Analytical Chemistry",
issn = "0003-2700",
publisher = "American Chemical Society",
number = "21",

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Phosphotyrosine biased enrichment of tryptic peptides from cancer cells by combining pY-MIP and TiO2 affinity resins. / Bllaci, Loreta; Torsetnes, Silje Bøen; Wierzbicka, Celina Katarzyna; Shinde, Sudhirkumar; Sellergren, Börje; Rogowska-Wrzesinska, Adelina; Jensen, Ole Nørregaard.

I: Analytical Chemistry, Bind 89, Nr. 21, 2017, s. 11332–11340.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - Phosphotyrosine biased enrichment of tryptic peptides from cancer cells by combining pY-MIP and TiO2 affinity resins

AU - Bllaci, Loreta

AU - Torsetnes, Silje Bøen

AU - Wierzbicka, Celina Katarzyna

AU - Shinde, Sudhirkumar

AU - Sellergren, Börje

AU - Rogowska-Wrzesinska, Adelina

AU - Jensen, Ole Nørregaard

PY - 2017

Y1 - 2017

N2 - Protein phosphorylation at distinct tyrosine residues (pY) is essential for fast, specific and accurate signal transduction in cells. Enrichment of pY-containing peptides derived from phosphoproteins is commonly facilitated by use of immobilized anti-pY antibodies prior to phosphoproteomics analysis by mass spectrometry. We here report on an alternative approach for pY-peptide enrichment using inexpensive pY imprinted polymer (pY-MIP). We assessed by mass spectrometry the performance of pY-MIP for enrichment and sequencing of phosphopeptides obtained by tryptic digestion of protein extracts from HeLa cells. The combination of pY-MIP and TiO2 based phosphopeptide enrichment provided more than 90% selectivity for phosphopeptides. Mass spectrometry signal intensities were enhanced for most pY-phosphopeptides (approximately 70%) when using the pY-MIP-TiO2 combination as compared to TiO2 alone. pY constituted up to 8% of the pY-MIP-TiO2 enriched phosphopeptide fractions. The pY-MIP-TiO2 and the TiO2 protocols yielded comparable numbers of distinct phosphopeptides, 1693 and 1842 respectively, from microgram levels of peptide samples. Detailed analysis of physicochemical properties of pY-MIP-TiO2 enriched phosphopeptides demonstrated that this protocol retrieved phosphopeptides that tend to be smaller (<24 residues), less acidic and almost exclusively monophosphorylated, as compared to TiO2 alone. These unique properties render the pY-MIP based phosphopeptide enrichment technique an attractive alternative for applications in phosphoproteomics research.

AB - Protein phosphorylation at distinct tyrosine residues (pY) is essential for fast, specific and accurate signal transduction in cells. Enrichment of pY-containing peptides derived from phosphoproteins is commonly facilitated by use of immobilized anti-pY antibodies prior to phosphoproteomics analysis by mass spectrometry. We here report on an alternative approach for pY-peptide enrichment using inexpensive pY imprinted polymer (pY-MIP). We assessed by mass spectrometry the performance of pY-MIP for enrichment and sequencing of phosphopeptides obtained by tryptic digestion of protein extracts from HeLa cells. The combination of pY-MIP and TiO2 based phosphopeptide enrichment provided more than 90% selectivity for phosphopeptides. Mass spectrometry signal intensities were enhanced for most pY-phosphopeptides (approximately 70%) when using the pY-MIP-TiO2 combination as compared to TiO2 alone. pY constituted up to 8% of the pY-MIP-TiO2 enriched phosphopeptide fractions. The pY-MIP-TiO2 and the TiO2 protocols yielded comparable numbers of distinct phosphopeptides, 1693 and 1842 respectively, from microgram levels of peptide samples. Detailed analysis of physicochemical properties of pY-MIP-TiO2 enriched phosphopeptides demonstrated that this protocol retrieved phosphopeptides that tend to be smaller (<24 residues), less acidic and almost exclusively monophosphorylated, as compared to TiO2 alone. These unique properties render the pY-MIP based phosphopeptide enrichment technique an attractive alternative for applications in phosphoproteomics research.

U2 - 10.1021/acs.analchem.7b02091

DO - 10.1021/acs.analchem.7b02091

M3 - Journal article

VL - 89

SP - 11332

EP - 11340

JO - Analytical Chemistry

JF - Analytical Chemistry

SN - 0003-2700

IS - 21

ER -