Real-time fluorescence monitoring of tryptic digestion in proteomics

Peter Karuso, Angela S. Crawford, Duncan A. Veal, G. B I Scott, Hung Yoon Choi

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Ensuring that proteolytic digestions are complete before submitting samples for downstream proteomic analyses is important, as failure or partial digestion can waste valuable instrument time and make results difficult to interpret. Conversely, overdigestion can also be problematic, such as when removing affinity tags from recombinant proteins or using nonspecific proteases. The techniques of HPLC, circular dichroism, SDS-PAGE, and MS have each been used to assess protein digestion. These techniques are slow, may require expensive instrumentation, can be inaccurate, and/or are unsuitable for real-time monitoring. Epicocconone is a natural fluorophore that reacts reversibly with proteins to form a highly fluorescent adduct and has previously been used to quantify proteins in 1D and 2D gels and in solution. Here, we describe a new method for the real-time monitoring of protein digestion based on epicocconone. This unique in situ fluorescent assay can tracelessly follow proteolysis of samples, at low microgram levels, destined for proteomics analysis or purification.

LanguageEnglish
Pages361-366
Number of pages6
JournalJournal of Proteome Research
Volume7
Issue number1
DOIs
Publication statusPublished - Jan 2008

Fingerprint

Proteomics
Proteolysis
Digestion
Fluorescence
Monitoring
Proteins
Circular Dichroism
Recombinant Proteins
Fluorophores
Polyacrylamide Gel Electrophoresis
Peptide Hydrolases
Gels
High Pressure Liquid Chromatography
Purification
Assays
epicocconone

Cite this

Karuso, Peter ; Crawford, Angela S. ; Veal, Duncan A. ; Scott, G. B I ; Choi, Hung Yoon. / Real-time fluorescence monitoring of tryptic digestion in proteomics. In: Journal of Proteome Research. 2008 ; Vol. 7, No. 1. pp. 361-366.
@article{8c6d3b9157bd48f79455503ba5e3e4b4,
title = "Real-time fluorescence monitoring of tryptic digestion in proteomics",
abstract = "Ensuring that proteolytic digestions are complete before submitting samples for downstream proteomic analyses is important, as failure or partial digestion can waste valuable instrument time and make results difficult to interpret. Conversely, overdigestion can also be problematic, such as when removing affinity tags from recombinant proteins or using nonspecific proteases. The techniques of HPLC, circular dichroism, SDS-PAGE, and MS have each been used to assess protein digestion. These techniques are slow, may require expensive instrumentation, can be inaccurate, and/or are unsuitable for real-time monitoring. Epicocconone is a natural fluorophore that reacts reversibly with proteins to form a highly fluorescent adduct and has previously been used to quantify proteins in 1D and 2D gels and in solution. Here, we describe a new method for the real-time monitoring of protein digestion based on epicocconone. This unique in situ fluorescent assay can tracelessly follow proteolysis of samples, at low microgram levels, destined for proteomics analysis or purification.",
author = "Peter Karuso and Crawford, {Angela S.} and Veal, {Duncan A.} and Scott, {G. B I} and Choi, {Hung Yoon}",
year = "2008",
month = "1",
doi = "10.1021/pr0704480",
language = "English",
volume = "7",
pages = "361--366",
journal = "Journal of Proteome Research",
issn = "1535-3893",
publisher = "AMER CHEMICAL SOC",
number = "1",

}

Real-time fluorescence monitoring of tryptic digestion in proteomics. / Karuso, Peter; Crawford, Angela S.; Veal, Duncan A.; Scott, G. B I; Choi, Hung Yoon.

In: Journal of Proteome Research, Vol. 7, No. 1, 01.2008, p. 361-366.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Real-time fluorescence monitoring of tryptic digestion in proteomics

AU - Karuso, Peter

AU - Crawford, Angela S.

AU - Veal, Duncan A.

AU - Scott, G. B I

AU - Choi, Hung Yoon

PY - 2008/1

Y1 - 2008/1

N2 - Ensuring that proteolytic digestions are complete before submitting samples for downstream proteomic analyses is important, as failure or partial digestion can waste valuable instrument time and make results difficult to interpret. Conversely, overdigestion can also be problematic, such as when removing affinity tags from recombinant proteins or using nonspecific proteases. The techniques of HPLC, circular dichroism, SDS-PAGE, and MS have each been used to assess protein digestion. These techniques are slow, may require expensive instrumentation, can be inaccurate, and/or are unsuitable for real-time monitoring. Epicocconone is a natural fluorophore that reacts reversibly with proteins to form a highly fluorescent adduct and has previously been used to quantify proteins in 1D and 2D gels and in solution. Here, we describe a new method for the real-time monitoring of protein digestion based on epicocconone. This unique in situ fluorescent assay can tracelessly follow proteolysis of samples, at low microgram levels, destined for proteomics analysis or purification.

AB - Ensuring that proteolytic digestions are complete before submitting samples for downstream proteomic analyses is important, as failure or partial digestion can waste valuable instrument time and make results difficult to interpret. Conversely, overdigestion can also be problematic, such as when removing affinity tags from recombinant proteins or using nonspecific proteases. The techniques of HPLC, circular dichroism, SDS-PAGE, and MS have each been used to assess protein digestion. These techniques are slow, may require expensive instrumentation, can be inaccurate, and/or are unsuitable for real-time monitoring. Epicocconone is a natural fluorophore that reacts reversibly with proteins to form a highly fluorescent adduct and has previously been used to quantify proteins in 1D and 2D gels and in solution. Here, we describe a new method for the real-time monitoring of protein digestion based on epicocconone. This unique in situ fluorescent assay can tracelessly follow proteolysis of samples, at low microgram levels, destined for proteomics analysis or purification.

UR - http://www.scopus.com/inward/record.url?scp=38649104430&partnerID=8YFLogxK

U2 - 10.1021/pr0704480

DO - 10.1021/pr0704480

M3 - Article

VL - 7

SP - 361

EP - 366

JO - Journal of Proteome Research

T2 - Journal of Proteome Research

JF - Journal of Proteome Research

SN - 1535-3893

IS - 1

ER -