CPSA Digest 2002

Proceedings -Tuesday, October 8, 2002

TuOD2

Isotope Coded Reduction Off of Chromatographic Support

Background
Cells exist in multiple states depending on normal or altered function. One way to compare the dissimilarity between two states of a cell is to quantify differences in the kinds and amounts of proteins present in these states. However, a major problem in protein analysis is the quantitative comparison of two proteins in different cellular sites that also vary in abundance.

One successful method for the quantitation of differential protein expression commonly uses protein-labeling with Isotope Coded Affinity Tag (ICAT) reagents and relies on mass spectrometry for analysis of the isolated and tagged peptides. This method permits the identification of proteins within a mixture and the quantitation of the difference in abundance for two proteins present in two or more samples.

The ICAT reagent consists of three components:

1. a thiol-specific reactive group that binds to the side chain of reduced cysteines
2. a deuterated or nondeuterated ethylene glycol linker group, and
3. a biotin tag used for affinity isolation of the ICAT-labeled peptides from complex mixtures using Avidin affinity chromatography.

The procedure for use of the ICAT reagent follows. The reduced cysteine residues of the proteins in two samples to be compared are labeled with either the heavy or light (normal) isotope tag. The two samples are added together, digested with trypsin, and then Avidin affinity chromatography is used to isolate the tagged peptides. Analysis by mass spectrometry provides information about the amino acid sequence and the relative abundance of each protein in the two samples. The quantitative information is derived from measuring and comparing the relative signal intensities of the pair of chemically identical peptides labeled with the light and heavy forms of the ICAT in the MS mode. The ratio of the peptide pairs provides quantitative information on the peptide of interest. Information on peptide identification is obtained by switching to the MS/MS mode and the spectra are used for peptide sequence determination. Databases are searched and matched against available sequences for protein identification.

Premise
The methods for relative protein quantitation are evolving and one example is the use of Isotope Coded Reducible Affinity Proteomics (ICRAP). Here, the tag does not significantly alter the ionization and fragmentation characteristics of the cysteine-containing peptides. The ICRAP approach uses a biotinylation reagent (Biotin-HPDP) containing a reducible linker that reacts with cysteine-containing peptides via a reducible disulfide bond. Use of the reducible linker allows for cleaner sample preparations since gentle elution conditions can be used.

The biotinylated peptides are isolated on an Avidin column and then they are released by reduction using dithiothreitol. These released peptides are then alkylated with either N-ethyl-iodoacetamide or N-ethyl-d5-iodoacetamide to prevent formation of mixed disulfide bonds; differentially labeled samples are mixed and analyzed by mass spectrometry. The alkylating reagents are synthesized from ethyl-amine (or ethyl-d5-amine) and iodoacetic anhydride.

This approach was successfully used to identify additional substrates of cell surface metalloproteases involved in shedding in response to LPS and phorbol ester stimulation. Two mixtures of proteins were prepared, each containing the same components and differing only in relative quantities. The two mixtures were worked up separately, tagged with one of the two reagents (D0 or D5 N-ethyl iodoacetamide) and then combined. The expected molar ratios for each protein were compared to what was observed from the peak height ratios. The procedure for Isotope Coded Reduction Off Chromatographic Support (ICROC) is shown below.

The absence of non-specifically binding (non-cysteine containing) peptides was discussed. The effect of the isotope tag on LC elution, peptide ion charge, and tandem mass spectral fragmentation was also discussed. The advantages of ICROC/ICRAP vs. ICAT were described as:

• Less non-specific peptide binding
• Reduced mass of isotope tag
• No change in peptide ion charge states
• No abundant tag-specific fragment ions
• Inexpensive reagents

Some issues in the comparison of ICROC vs. ICRAP are: ICROC does not need to use HPDP-biotin, which has limited solubility; there are no Avidin tryptic peptide background ions; and ICROC is cheaper (no Avidin or biotinylation reagents) and simpler. However, ICROC has a few difficulties, namely that sample preparation and data acquisition are lengthy (but can be automated); data interpretation is currently a manual process, and there are no archiving or database tools available for quantitative information.

ICROC is the favored method in this laboratory for cysteine-specific isotope labeling for these reasons:

• less non-specific binding
• lower mass Cys label that does not change the charge state
• no label-specific fragmentation observed

References
S.P. Gygi et al., "Quantitative analysis of complex protein mixtures using isotope coded affinity tags." Nature Biotechnology 17 (1999) 994-999.

A.J. Link et al., "Direct analysis of protein complexes using mass spectrometry." Nature Biotechnology 17 (1999) 676-682.

Richard S. Johnson, Alison Wallace, Lin Guo and Min Shen, "Isotope Coded Reducible Affinity Proteomics (ICRAP)," Proceedings 49th American Society for Mass Spectrometry Conference, Chicago, IL USA (2001).

Min Shen, Richard S. Johnson and Alison Wallace, "Isotope Coded Reducible Affinity Phosphate Detection In Peptides Involving Ser/Thr (ICRAPDINPIST)." Proceedings 49th American Society for Mass Spectrometry Conference, Chicago, IL USA (2001).

John M. Peltier, Sofia G. Hohnholt, Xiangping Zhu, Jon DeGnore and Edward J. Takach, "Quantitation and Identification of Proteins using Isotope Coded Affinity Tags: A Comparison of MALDI-TOF and ESI-TOF Mass Spectrometry." Proceedings 49th American Society for Mass Spectrometry Conference, Chicago, IL USA (2001).

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