Molecular Probes

Section 9.5 — Reagents for Peptide Analysis, Sequencing and Synthesis

This section describes Molecular Probes' reagents used in the synthesis of fluorescent dye– or hapten-labeled peptides and fluorogenic protease substrates, as well as in peptide and protein sequencing. The dominant chemistry for sequencing peptides employs the nonfluorescent reagent phenyl isothiocyanate, which forms phenylthiohydantoins (PTH) in the sequencing reaction. Some of our fluorescent probes and research chemicals have been used for N-terminal amino acid analysis and peptide sequencing, as well as for protein fragment modification prior to PTH sequencing.

N-Terminal Amino Acid Analysis

Except when it is already blocked by formylation, acetylation, pyroglutamic acid formation or other chemistry, the N-terminal amino acid of proteins can be labeled with a variety of fluorescent and chromophoric reagents from Chapter 1. However, only those functional groups that survive complete protein hydrolysis, such as sulfonamides, are useful for N-terminal amino acid analysis. Dansyl chloride (D21) and dabsyl chloride (D1537) are the most commonly employed reagents for such analyses.

Nonacylated N-terminal serine and threonine residues of proteins can be periodate-oxidized to aldehydes (Figure 3.1) that can then be modified by a variety of Hydrazine Derivatives and Hydroxylamine Derivatives listed in Section 3.2. Only peptides and proteins that contain these two terminal amino acids become fluorescent, although oxidation of the carbohydrate portion of glycoproteins to aldehydes may cause interference in this analysis.

N-Acetylated or N-formylated proteins have been detected by transfer of the acyl group to dansyl hydrazine (D100) and subsequent chromatographic separation of the fluorescent product. The sensitivity of this method can likely be improved by the use of other fluorescent Hydrazine Derivatives and Hydroxylamine Derivatives described in Section 3.2.

Peptide Sequencing

As analogs of phenyl isothiocyanate, the peptide conjugates of fluorescein-5-isothiocyanate (FITC; F143, F1906, F1907; Section 1.5) and other fluorescent Isothiocyanates are susceptible to Edman degradation via their thiohydantoins. Thus, these fluorescent reagents are potentially useful for ultrasensitive amino acid sequencing.

Peptide Synthesis

Peptides specifically labeled with fluorescent dyes, haptens, photoactive groups or radioisotopes are important both as probes for receptors and as substrates for enzymes (Section 10.4). Labeled peptides can be prepared by modifying isolated peptides or by incorporating the label during solid-phase synthesis. Molecular Probes offers some fluorescent neuropeptides, most of which are described in Section 16.2.

Labeling Peptides in Solution

Appropriately substituted synthetic peptides can be labeled in solution by almost any of the reactive probes in Chapters 1–5 (Technical Focus: Labeling Small Peptides with Amine-Reactive Dyes in Organic Solvents). Many peptides contain multiple residues that can be modified, potentially leading to complex mixtures of products, some of which may be biologically inactive. Modification of a peptide's thiol group by one of the thiol-reactive reagents described in Chapter 2 is usually easy, selective and very efficient. If the peptide is synthetic, or can be modified by site-directed mutagenesis, incorporation of a cysteine residue at the desired site of labeling is recommended. The N-terminus of peptides, which has a lower pK_a than the ε-amino group of lysine residues, can sometimes be labeled in the presence of other amines if the pH is kept near neutral. Conversion of tyrosine residues to o-aminotyrosines (Section 3.1, Figure 3.3) can be used to provide selective sites for peptide modification, unless the tyrosine residues are essential for the biological activity of the peptide.

Solid-Phase Synthesis of Labeled Peptides

Because specific labeling of peptides in solution is problematic, it may be more convenient to conjugate the fluorophore to the N-terminus of a resin-bound peptide before removal of other protecting groups and release of the labeled peptide from the resin. About five equivalents of an amine-reactive fluorophore are usually used per amine of the immobilized peptide. The fluorescein, eosin, Alexa Fluor, Oregon Green, Rhodamine Green, tetramethylrhodamine, Rhodamine Red, Texas Red, coumarin and NBD fluorophores, the QSY, dabcyl and dabsyl chromophores and biotin are all expected to be reasonably stable to hydrogen fluoride (HF) as well as to most other acids. These fluorophores, chromophores and biotin are also expected to be stable to reagents used for deprotection of peptides synthesized using FMOC chemistry. The BODIPY fluorophore may be unstable to the conditions used to remove some protecting groups.

Molecular Probes has prepared some unique reagents for automated synthesis of peptides that are specifically labeled with fluorophores, chromophores and haptens. Use of these precursors permits the incorporation of these groups at specific sites in the peptide's sequence. The α-FMOC derivative of ε-dabcyl-L-lysine (D6216) can be used to incorporate the dabcyl chromophore at selected sites in the peptide sequence. The dabcyl chromophore, which has broad visible absorption (Figure 10.55), has been extensively used as a quenching group in the automated synthesis of HIV protease (H2930, Section 10.4), renin (R2931, Section 10.4) and other fluorogenic peptidase substrates. The dabcyl group can also be incorporated at the N-terminus by using dabcyl succinimidyl ester (D2245). The aminonaphthalene derivative EDANS (A91) has been the most common fluorophore for pairing with the dabcyl quencher in fluorescence resonance energy transfer (FRET) experiments because its fluorescence emission spectrum overlaps the absorption spectrum of dabcyl (Figure 10.55) (Technical Focus: Fluorescence Resonance Energy Transfer (FRET)). This fluorophore is conveniently introduced during automated synthesis of peptides by using γ-EDANS-α-FMOC-L-glutamic acid (F11831) or the corresponding t-BOC derivative (B6215). The tetramethylrhodamine fluorophores can be incorporated during automated FMOC synthesis of peptides using our single-isomer α-(FMOC)-ε-TMR-L-lysine building block (F11830). Site-selective biotinylation of peptides can be achieved using the FMOC derivative of biocytin (B20651) during automated synthesis. This reagent can also be attached to the synthesis resin as the first residue to provide automated synthesis of C-terminal biotinylated peptides.

Our QSY dyes (Section 1.6, Section 1.8) have broad visible to near-infrared absorption (Table 1.10, Figure 1.70). These dyes, which are essentially nonfluorescent, are particularly useful as energy acceptors from blue-, green-, orange- or red-fluorescent donor dyes (Table 1.11). The QSY 7, QSY 9, QSY 21 and QSY 35 chromophores can be conjugated to amines via their succinimidyl esters (Q10193, Q20131, Q20132, Q20133). The QSY 7 dye can also be conjugated to thiols of peptides or to thiol-modified oligonucleotides via its maleimide (Q10257) and the QSY 35 dye coupled via its iodoacetamide (Q20348). Additionally, peptide amides can be prepared from the QSY 7 and QSY 35 aliphatic amines (Q10464, Q20540). We have also prepared α-(FMOC)-ε-QSY 7-L-lysine and α-FMOC-β-QSY 35-L-alanine (Q21930, Q21931), which can be used in the automated synthesis of QSY 7 quencher– or QSY 35 quencher–containing peptides.