Fluorescent labels for proteomics and genomics

Fluorescent labels for proteomics and genomicsAlan Waggoner

Fluorescent labeling reagents are an essential component of a

huge industry built on sensitive fluorescence detection. This

technology has grown over 30 years and is in some ways

mature. Excellent labeling reagents with close to maximum

theoretical brightness are available in many different colors.

Large fluorescent proteins like phycobiliproteins are also widely

used that are exceedingly bright. Other fluorescent proteins like

the GFP family can be obtained for creating genetically

encoded protein labels in living cells. A new ‘solid state’

quantum dot technology is being exploited for large-scale

multiparameter labeling. This technology provides the

‘ultimate’ photostable labeling reagent. Still, there are

advances to be made. Not available is the ultimate tool kit of

low molecular weight, strongly light absorbing, photostable

labels with narrow emission bands ranging from the UV to

the IR.

Addresses

Department of Biological Sciences, Carnegie Mellon University, 4400 5th

Avenue, Pittsburgh, PA 15213, USA

Corresponding author: Waggoner, Alan ([email protected])

Current Opinion in Chemical Biology 2006, 10:62–66

This review comes from a themed issue on

Proteomics and genomics

Edited by Garry P Nolan and Emanuel F Petricoin

Available online 18th January 2006

1367-5931/$ – see front matter

# 2006 Elsevier Ltd. All rights reserved.

DOI 10.1016/j.cbpa.2006.01.005

IntroductionProteomics and genomics often require labeling of com-

ponents for detection. Isotopes, enzyme-linked chromo-

phore/fluorophore production, chemiluminescence and

bioluminescence are among the methods that might be

considered. Mass spectrometry offers a label-less method

of detection. With these methods available, why have

fluorescent labels so often been the choice to the point

that fluorescence detection is the key in a multi-billion

dollar detection industry?

The answer is complex, and factors vary for different

applications. But generally the following characteristics

drive the decision:

1. F

Cu

ast signal acquisition: each individual fluorescent

label can potentially provide 107–108 photons per

second for detection.

rrent Opinion in Chemical Biology 2006, 10:62–66

2. M
ulti-colored dyes can be used for multiplex assays.
For example the four different bases in DNA

sequencing.

3. S
ensitivity: single molecule detection is becoming
prevalent for some applications.

4. S
mall label size means that there is little perturbation
of the behavior of the labeled material.

5. T
he signal is localized, unlike with some enzyme-
linked amplification schemes.

6. T
he labeling reagents are stable and robust for most
applications.

7. T
he labeling process is straightforward provided that
appropriate functional groups are available on the

target.

There are also limitations of fluorescence detection and

these are discussed below.

Early fluorescent labelsThe most widely used fluorescent labels are based on the

xanthene dyes or the cyanine structure (Figure 1).

Fluorescein was the first label (for immunofluorescence in

1953), with rhodamine not long after. Fluorescein is still

widely used despite certain disadvantages (photobleach-

ing and pH sensitivity). Rhodamines are pH insensitive

and more photostable than fluorescein analogs but are

more difficult to use because they possess a hydrophobic

planar structure that leads to low water solubility, non-

specific binding of labeled species and quenching of

fluorescence on labeled proteins because of dimerization

of boundmultiple rhodamine labels. The original reactive

groups on rhodamines and fluoresceins were isothiocya-

nates that react with free amino groups on proteins and

modified nucleic acids during the labeling (conjugation)

process. Sulfonyl chlorides can also be used for labeling

amino groups but the reaction is sometimes difficult to

control. Succinimidyl esters have become the preferred

reactive group for labeling amino groups on macromole-

cules. The reactions are easy to control and the linkage is

through a peptide bond. Maleimide and iodoacetamide

derivatives have been the mainstays for labeling sulfhy-

dryl groups.

Development of multicolor labelsIn the 1980s and early 1990s, efforts began to develop new

multicolor fluorescent labels for antibodies and DNA

probes to match advances in multiparameter flow and

image cytometry that were taking place. Phycoerythrin

was introduced in 1982 [1]. This powerful multi-chromo-

phore protein-labeling reagent obtained from photosyn-

thetic bacteria changed the flow cytometry industry and

www.sciencedirect.com

Fluorescent labels for proteomics and genomics Waggoner 63

Figure 1

Xanthene dyes and cyanine dye structures. (a) Rhodamine, (b)

fluorescein and (c) a cyanine. Each R-group is placed on the

fluorophore structure to do one of the following: (1) finely tune the

wavelength of absorption and emission; (2) control water solubility,

dimerization and non-specific binding; and (3) attach a reactive group

for labeling. In the cyanine dye ‘n’ controls the number of double bonds

in the structure and therefore provides for coarse tuning of the

absorption and fluorescence wavelengths.

permitted sensitive two-color lymphocyte subset analysis

with a single argon ion laser. Phycoerythrin excites very

strongly at 488 nm but the emission is shifted to 580 nm

by energy transfer events between chromophores within

the protein. Phcoerythrin as a second color works well

with fluorescein-labeled antibodies, can also be excited at

488 nm but are detected separately at 525 nm to provide

the second color of detection. HIV monitoring and cancer

diagnostics were a strong driving force for the growth of

this reagent. Three and four-color analysis with a single

laser are now commonplace with phycobiliprotein-based

energy transfer reagents. Low molecular weight fluores-

cent labels such as Cy5 and Cy7 (see below) are good

acceptors from excited PE and shift emission farther to


red and deep red wavelengths and 11-color analyses are

now possible [2�].

PE is also a very important reagent in proteomics and

genomics and is the basis of the detection system in

Affymetrix chips. PE-labeled streptavidin is added after

sample binding is complete and produces a strong signal

from array elements containing the biotin-labeled DNA

or protein probes. In this application, and in immuno-

phenotyping lymphocytes and cancer cells by flow cyto-

metry, the targets of phycoerythrin binding are easily

accessible to the bathing solution. However, the phyco-

biliproteins have drawbacks in that their size (�1–1.5

times the molecular weight of IgG) limits diffusion of

labeled proteins, streptavidin, antibodies or DNA probes

into regions of cells, tissues, chromosomes and gels where

the structural matrix is tight. Low molecular weight

organic dye fluorescent labels are �100 times smaller

in weight. Furthermore, they have other advantages. It

is easier to design and synthesize fluorescent dyes with

desirable excitation and emission wavelengths tuned to

the excitation sources of the fluorescence signal readout

instrument. A wider variety of functional groups are

available for conjugation. Conjugation can be very simple.

The charge and the solubility of the organic dyes can be

controlled to some extent with dye surface modification

groups. Sometimes, multiple low molecular weight dyes

can be attached to the detection reagent so that the

fluorescence signal is brighter, although it is difficult to

meet the brightness of a single phycoerythrin label.

Multicolor cyanine dye labels became commercially

available as labeling reagents in the early 1990s. Cy3,

Cy5, Cy7 and intermediate wavelength dyes were sold as

succinimidyl esters and appeared on a wide range of

immunological reagents for imaging, flow cytometry

and DNA probes for chromosome mutation analysis

[3]. The breakthrough was addition of negatively charged

sulfonate groups [4] directly to the ring systems of

brightly fluorescent cyanine dyes that were developed

originally as sensitizers for the photographic industry.

The sulfonates greatly improve water solubility and

reduce fluorescence-quenching dye–dye interactions that

can take place on the surface of antibodies. Cy3 and Cy5

have become widely used for labeling RNA and DNA for

gene expression studies [5]. Unlike some reagents, they

remain fluorescent when the microarray is dry, making

array readout more robust.

An interesting example of fluorophore engineering to

produce a finely tuned proteomics detection system

can be found in DIGE (differential gel electrophoresis).

This system was developed by Minden [6] and is now

marketed by GE Healthcare. The goal of the develop-

ment was to get around the use of two separate gels when

comparing a protein profile from sample cells with the

‘unperturbed’ profile from control cells. The approach


64 Proteomics and genomics

Figure 2

Schematic of the process of Difference gel electrophoresis (DIGE).

Courtesy of Jon Minden, Carnegie Mellon University.

was to create two different color fluorescent protein labels

that have identical molecular weight and that react with

amino groups on proteins without changing the charge of

the labeled lysine residue. Cy3 and Cy5 analogs were

synthesized. Cy3 is used to label extracted test protein

and Cy5 is the label for extracted control protein. After

extraction and labeling, they are mixed and applied to the

same 2-D SDS gel for electrophoresis (Figure 2). The two

different color images can be merged or ratioed to quan-

tify changes in the protein profile. After fluorescence

imaging and analysis, an automated spot picker can isolate

the different protein spots for mass spectroscopy [7,8].

The ABI ‘big dyes’ have dominated DNA sequencing

technology for over a decade. These are a carefully

engineered set of fluorescein derivatives that allow

four-color sequencing in gels and capillary array readout

systems. The four labels constructed from analogs of

FAM, JOE, ROX and TAMRA can be excited at 488

nm but the four reagents emit at different wavelengths

[9]. For shifting to longer wavelengths, energy-transfer

complexes were synthesized. Although the molecular

weight of the energy transfer labels on the DNA termi-

nators was more than double from that of a single dye, the

system and software were engineered so that > 500

sequences can be read in a single experiment. The system

incorporates sophisticated software to compensate for

spill over of signals from different labels into detection

channels of other labels. However, even now multicolor

detection technology such as DNA sequencing would

significantly benefit from sets of wavelength shift labels

that have narrow emission bands and minimal spillover of

signals. Quantum dots (see below) are exceptional in this

advantage, but their large size appears to preclude use in


detection systems that measure mobility. Incorporation of

multiple excitation sources with other low molecular

weight dyes such as the cyanines, BODIPYs, xanthenes

and ‘Big Dyes’ into high-throughput, low cost DNA

sequencing systems is under development [10].

Alexa fluorescent labels developed by Richard Haugland

and his team at Molecular probes (now marketed by

Invitrogen) adapt the concept of aryl-sulfonation that

made the cyanine dyes useful as labels. The new Alexa

dyes emit from 442 nm to 775 nm. GE Healthcare sells

CyDye reagents and several other companies including

Pierce offer new additions to the range of low molecular

weight labels.

There has been a noticeable trend to develop and use

new labeling reagents that fluoresce at longer wave-

lengths. There are advantages. More parameters can be

measured with sets of dyes that include longer wave-

lengths. However, the most significant advantage is the

reduced background fluorescence from cells, cell debris,

buffer components and plastic materials at long wave-

lengths. Also, when these labels were originally devel-

oped they could be excited by inexpensive red-emitting

lasers and laser diodes. Inexpensive solid state excitation

sources in the blue and green were not available then as

they are now. Patonay and Licor were among the pioneers

in developing and incorporating infrared cyanine dye

labels for DNA sequencing [11]. They continue tomarket

a range of IR cyanine dyes for genomics and proteomics.

Infrared fluorescent labels have the disadvantage of lower

chemical and photostability. A new generation of more-

stable low molecular weight IR fluorescing organic dyes

would be welcomed for development of sensitive detec-

tion systems.

The lanthanide chelate labels have been a tempting

alternative to pure organic labels for several decades

but have not been adapted for large-scale genomics or

proteomics. The ability to improve signal-to-noise

through time-resolved detection of the lanthanide

micro-millisecond emission decay is appealing. However,

there are limitations that remain with these reagents.

Extinction coefficients of lanthanide chelates are not

generally large, their quantum yields are modest at best,

and they have lower duty cycles for excitation and emis-

sion due to their long lifetimes. Nevertheless, continuing

innovations in chelation chemistry for lanthanides may

hold promise [12].

Are larger fluorescent labels likely to be usefulin proteomics and genomics?We have pointed out that phycoerythrin, despite its

molecular size, is a powerful detection reagent for certain

microarrays (but not gels and electrophoresis systems).

What else is available? The first relatively new technology

that comes to mind is the solid-state nanoparticles or


Fluorescent labels for proteomics and genomics Waggoner 65

Figure 3

Absorption and emission spectra of selected organic dyes and quantum dots. (a) Absorption (left)–emission (right) pairs of the spectra of five

organic fluorophores. These spectra illustrate the spillover of absorption and emission wavelengths into the regions where other dyes absorb and

emit. Except for DAPI, these fluorophores represent organic dyes with relatively narrow band spectra. (b) Absorption and emission spectra of

three different quantum dots [13]. Note the narrow emission bands with lack of spectral spillover. Notice also that all three quantum dots samples

can be efficiently excited in the blue region of the spectrum.

quantum dots recently reviewed in this publication by

Bruchez [13]. Quantum dots have great advantages. They

fluoresce throughout the visible and near infrared and can

be excited very efficiently with one blue (uv) excitation

source (Figure 3b). They also have narrow emission bands

so that, in principle, as many as 10–20 quantum dot

reagents could be detected separately with narrow band-


pass filters. If properly coated, quantum dots have quan-

tum yields approaching 0.9. The challenge has been

achieving proper coating to preserve stability, water solu-

bility and capability of labeling targets. A great deal of

progress has been made on this front and reagent sets

should eventually be effective for multiparameter detec-

tion with microarray devices.


66 Proteomics and genomics

Another type of large fluorophore labeling for proteomics

that we have not covered is the labeling of proteins with

green fluorescent protein (GFP). High-throughput auto-

mated sub-cellular characterization of the proteome is

being addressed by classification of distribution patterns

of GFP-labeled proteins by imaging microscopy. This

may open a wide range of applications including large-

scale RNAi screening in mammalian cells [14,15].

Are the existing labels good enough?We have mentioned a wide range of applications of low

molecular weight (and also large) fluorescent labels.

These include applications in sequencing, gene expres-

sion, protein gel analysis and protein and nucleic acid

microarrays. With these high-impact successes, do we

need improved or additional fluorophores?

The existing fluorophores are brightly fluorescent.

Excluding effects of local environment, brightness is

proportional to the extinction coefficient (at l-excitation)

times the quantum yield (assuming a significant part of

the emission spectrum is detected). Those dyes in the

visible region of the spectrum have extinction coefficients

in the 70–250 kl/(mol-cm) range, which is about the

maximum predicted for the number of double bonds

and heteroatoms of these chromophores. The quantum

yields of the useful dyes range from 0.3 to 0.8. Thus, they

are within a small factor (�1.5–3) of having the maximum

fluorescence. There is little pressure in the fluorescence

detection industry for incremental improvement of

brightness, especially because complicated instrument

reagent systems are often not amenable to incrementally

improved dye because fine tuning of reagent packages

and optical components would have to be done again at

large expense. Photostability is also generally not a pro-

blem because readout is fast in high-throughput large-

scale systems.

Nevertheless, there are properties in need of improve-

ment. Chemical stability for robust sample preparation,

shipping, storage and sample manipulation offers an

avenue for improvement of IR labels. If the number of

parameters to be detected during readout is larger than

about four there will be challenges for the use of existing

organic labels (Figure 3a). Again quantum dots may offer

relief for this challenge; however, the size of the quantum

dots could still present problems for some assays. So far

(e.g. sequencing and DIGE), engineering of the fluoro-

phores and instrumentation optics and software has pro-

vided ways to work around the problem of spectral

spillover into other detection channels.

ConclusionsIt will be interesting to see what the new demands might

be for fluorophores as new nano-based high-throughput

systems evolve over upcoming decades. If there is con-


tinued movement toward obtaining data from single-

molecule fluorescence detection measurements, there

may be a need for fluorophores with increasing photo-

stability that can withstand the large photon fluxes

needed to quickly access the required number of

detected photons.

AcknowledgementsThis work was supported by NIH grants R33 CA97541 and R01EB00364.

References and recommended readingPapers of particular interest, published within the annual period ofreview, have been highlighted as:

� of special interest

�� of outstanding interest

1. Oi VT, Glazer AN, Stryer L: Fluorescent phycobiliproteinconjugates for analyses of cells and molecules. J Cell Biol1982, 93:981-986.

2.�

De Rosa SC, Brenchley JM, Roederer M: Beyond six colors: anew era in flow cytometry. Nat Med 2003, 9:112-117.

A great example of fluorescent probes and the power of multiparameterflow cytometry.

3. Kallioniemi A, Kallioniemi OP, Sudar D, Rutovitz D, Gray JW,Waldman F, Pinkel D: Comparative genomic hybridization: apowerful newmethod for cytogenetic anlaysis of solid tumors.Science 1992, 258:818-821.

4. Mujumdar RB, Ernst LA, Mujumdar SR, Lewis CJ, Waggoner AS:Cyanine dye labeling reagents: Sulfosuccinimidyl esters.Bioconjugate Chemistry 1993, 4:105-111.

5. Schena M, Shalon D, Davis RW, Brown PO: Quantitativemonitoring of gene expression patterns with a complementaryDNA microarray. Science 1995, 270:467-470.

6. Unlu M, Morgan ME, Minden JS: Difference gel electrophoresis:a single gel method for detecting changes in protein extracts.Electrophoresis 1997, 18:2071-2077.

7. Baldwin MA: Protein identification by mass spectrometry.Mol Cellular Proteomics 2004, 3.1:1-9.

8. Freman WM, Hemby SE: Proteomics for protein expressionprofiling in neuroscience. 2004; 29: 1065-1081.

9. Ju J, Ruan C, Fuller CW, Glazer AN, Mathies RA: Fluorescenceenergy transfer dye-labeled primers for DNA sequencing andanalysis. Proc Natl Acad Sci USA 1995, 92:4347-4351.

10. Lewis EK, Haaland WC, Nguyen F, Heller DA, Allen MJ,MacGregor RR, Bergere SC, Willingham B, Burns LA, Scott GBIet al.: Color-blind fluorescence detection of four-color DNAsequencing. Proc Natl Acad Sci USA 2005, 102:5346-5351.

11. Baars MJ, Patonay G: Interfacing a high-sensitivity, near-infrared laser-induced fluorescence detector with acommercial capillary electrophoresis instrument. ApplSpectrosc 1998, 52:619-622.

12. Zhang J, Badger PD, Geib SJ, Petoud S: Sensitization of near-infrared-emitting lanthanide cations in solution by tropolonateligands. Angew Chem Int Ed Engl 2005, 44:2508-2512.

13. Bruchez M: Turning the lights on: quantum dots in cellularassays. Curr Opin Chem Biol 2005, 9:533-537.

14. Conrad C, Erfle H, Warnat P, Daigle N, Lorch T, Ellenberg J,Pepperkok R, Eils R: Automated identification of subcellularphenotypes on human cell arrays. 2004; 14: 1130-1136.

15. Chen X, Velliste M, Weinstein S, Jarvik JW, Murphy RS: Locationproteomics: building subcellular location tree from highresolution 3D fluorescence microscope images of randomly-tagged proteins. Proc SPIE 2003, 4962:298-306.


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Fluorescent labels for proteomics and genomics