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Quantitative proteomics: assessing the spectrum of in-gel protein detection methods

  • Review
  • Published:
Journal of Chemical Biology

Abstract

Proteomics research relies heavily on visualization methods for detection of proteins separated by polyacrylamide gel electrophoresis. Commonly used staining approaches involve colorimetric dyes such as Coomassie Brilliant Blue, fluorescent dyes including Sypro Ruby, newly developed reactive fluorophores, as well as a plethora of others. The most desired characteristic in selecting one stain over another is sensitivity, but this is far from the only important parameter. This review evaluates protein detection methods in terms of their quantitative attributes, including limit of detection (i.e., sensitivity), linear dynamic range, inter-protein variability, capacity for spot detection after 2D gel electrophoresis, and compatibility with subsequent mass spectrometric analyses. Unfortunately, many of these quantitative criteria are not routinely or consistently addressed by most of the studies published to date. We would urge more rigorous routine characterization of stains and detection methodologies as a critical approach to systematically improving these critically important tools for quantitative proteomics. In addition, substantial improvements in detection technology, particularly over the last decade or so, emphasize the need to consider renewed characterization of existing stains; the quantitative stains we need, or at least the chemistries required for their future development, may well already exist.

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Abbreviations

2DE:

two-dimensional electrophoresis

BisANS:

4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid

BSA:

bovine serum albumin

C16-F:

C-16 fluorescein

CA:

carbonic anhydrase

CBB:

Coomassie Brilliant Blue

cCBB:

colloidal Coomassie Brilliant Blue

CCD:

cooled charge device

Cy2:

Cyanine 2

Cy3:

Cyanine 3

Cy5:

Cyanine 5

cys:

cysteine

DIGE:

differential gel electrophoresis

DMSO:

dimethyl sulfoxide

DP:

deep purple

DTT:

dithiothreitol

EBT:

erichrome black T

EDTA:

ethylene diamine tetraacetic acid

EtOH:

ethanol

EZ:

ethyl violet and zincon

HAc:

acetic acid

HSA:

human serum albumin

ICy:

iodoacetylated cyanine dye

IEF:

isoelectric focusing

LC:

liquid chromatography

LDR:

linear dynamic range

LLD:

lowest limit of detection

lys:

lysine

MALDI-ToF-MS:

matrix-assisted laser desorption ionization-time of flight-mass spectrometry

mBB:

monobromobimane

MDPF:

two-methoxy-2,4-diphenyl-3(2H)-furanone

MeOH:

methanol

MS:

mass spectrometry

MW:

molecular weight

OPA:

o-phthalaldehyde

OVA:

ovalbumin

PAGE:

polyacrylamide gel electrophoresis

PAS:

periodic acid-Schiff

PhosB:

phosphorylase b

PMF:

peptide mass fingerprinting

RuBPS:

ruthenium (II) tris (bathophenanthroline disulfonate)

SA:

Stains All (1-ethyl-2-{3- [1-ethylnaphtho (1,2d) thiazolin-2-ylidene]-2-methyl-propenyl}-naptho (1,2d) thiazolium bromide)

SDS:

sodium dodecyl sulfate

SNR:

signal-to-noise ratio

sp.:

species

SR:

SYPRO Ruby

TCEP:

Tris (2-carboxyethyl) phosphine hydrochloride

Trp:

tryptophan

UV:

ultraviolet

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Acknowledgements

The authors thank Dr. R.H. Butt for insightful discussions during the writing of this review.

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  1. Molecular Physiology, School of Medicine, and Molecular Medicine Research Group, University of Western Sydney, Campbelltown, NSW, 1797, Australia

    Victoria J. Gauci, Elise P. Wright & Jens R. Coorssen

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  1. Victoria J. Gauci
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  2. Elise P. Wright
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  3. Jens R. Coorssen
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Correspondence to Jens R. Coorssen.

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Victoria J. Gauci and Elise P. Wright contributed equally to this study.

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Gauci, V.J., Wright, E.P. & Coorssen, J.R. Quantitative proteomics: assessing the spectrum of in-gel protein detection methods. J Chem Biol 4, 3–29 (2011). https://doi.org/10.1007/s12154-010-0043-5

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