Fig. 1. The use of chemical tagging strategies for sample fractionation. A protein mixture is either first labeled with an affinity tag and then digested (left) or first digested and then labeled (right). In both cases, labeled peptides are subsequently enriched by an affinity chromatography step, so that ideally only the tagged peptides remain.

Fig. 2. Concept of stable-isotope labeling for relative protein quantitation. Two samples containing different protein amounts (red and blue, respectively) are digested seperately and the protein mixtures are then individually labeled by an isotope tag in either its light (white circles) or heavy form (grey circles). After combination of the two samples, further analysis is performed on the combined peptide pool. Mass spectra show signal pairs of the same intensity when equal protein amounts were originally present (bottom, left). Differences in abundance are reflected in a ratio other than one, in this case 2:1 (bottom, right). Alternative workflows are also possible.

Fig. 3. Simplification of MS/MS spectra by charge neutralization. Left: A model peptide ABBCD (with A, B, C and D representing different amino acids) is subjected to collision-induced dissociation, resulting in a mixture of different fragments, making sequence elucidation de novo difficult. Right: After the attachment of a permanent negative charge on the N-terminus (denoted by an asterisk), the formation of b-ions is suppressed and y-ions constitute most of the product ions, making sequence elucidation straightforward.

Fig. 4. Structure of the original isotope-coded affinity tag (ICAT) reagent [47]. The tag consists of a biotin moiety that allows enrichment by biotin–avidin affinity chromatography (A), an isotope-coded linker region, using hydrogen or deuterium in the first version (B), and a thiol-reactive iodoacetamide group that allows alkylation of cysteine residues with the ICAT (C).

Fig. 5. Schematic representation of the ICAT workflow. ICAT-labeling can also be performed after the digestion step, so that peptides, not intact proteins, are labeled (not shown). For details, see text.

Fig. 6. Improved isotope-coded affinity tags. (a) The solid-phase ICAT [69], including a photocleavable linker region. (b) The cleavable ICAT [67], [70], [71], [72], [73], [74], [75], [76] and [77] with an acid-labile affinity tag region. Asterisks denote differentially (¹²C/¹³C) labeled carbon atoms.

Fig. 7. Concept of the VICAT (visible isotope-coded affinity tag) strategy [78]. (a) Chemical structure of the VICAT reagents. (b) VICAT workflow. For details, see text.

Fig. 8. Thiol-specific reagents for differential isotope coding of cysteine residues (X = hydrogen or deuterium).

Fig. 9. Various cysteine-specific affinity tags. (a) ALICE solid-phase tag of Qiu et al. [99] (where X = H or D). (b) Solid-phase tag of Shi et al. [100]. Asterisks denote differentially (¹²C/¹³C) labeled carbon atoms. (c) Element-coded affinity tag of Whetstone et al. [102] (where M is a rare-earth metal).

Fig. 10. Principle of disulfide exchange covalent chromatography applied to the isolation of cysteine-containing peptides. For details, see text.

Fig. 11. Structure of the APTA tagging reagent introduced by Regnier and co-workers [106].

Fig. 12. Principle of the HysTag method [107]. Cysteine residues in proteins are differentially tagged with the peptidic tag (X = H or D) and digested with endoproteinase Lys-C. The (His)₆-sequence on the tag allows the enrichment of the tagged peptides by either immobilized metal affinity chromatography (IMAC) or strong cation exchange chromatography (SCX). A second digestion step, this time with trypsin, cleaves the tag C-terminal to arginine, so that only a Cys-Ala*-dipeptide containing the isotope tag (*) remains on the labeled cysteines.

Fig. 13. Concept of diagonal chromatography [109]. The crude sample is fractionated and a subset of the peptides contained in each fraction is tagged in a way so that their chromatographic behavior is altered. Upon reinjection, most of the peptides from the fractions elute at the same position as before, only the tagged peptides show different elution times (boxes marked with an asterisk) and can be collected for further characterization.

Fig. 14. Various lysine- and tryptophan-specific tags for relative quantification applications.

Fig. 15. The protein sequence tag strategy [122] and [123] for the isolation of N-terminal peptides from cyanogen bromide cleaved proteins. For details, see text.

Fig. 16. Isotope-coded tags for labeling of peptide N-termini and lysine amino groups (X = hydrogen or deuterium).

Fig. 17. Reaction scheme of the phosphopeptide tagging approach presented by Zhou et al. [167]. A phosphopeptide is shown in a simplified version highlighting its N- and C-terminus and the phosphate group of a phosphorylated residue within the peptide chain. For details, see text.

Fig. 18. Conecpt of phosphopeptide tagging using β-elimination of the phosphate group from pSer and pThr and attachment of various affinity tags.

Fig. 19. Phosphopeptide tagging procedure of McLachlin and Chait [175], including a β-elimination step and disulfide exchange chromatography to isolate DTT-labeled peptides. For details, see text.

Fig. 20. Identification strategies for N-linked glycoproteins in complex mixtures using solid-phase capture and hydrazide chemistry [190] (left) or tandem lectin affinity chromatography [186] (right), both in combination with differential isotope coding for relative quantitation.

Fig. 21. Periodate oxidation of cis-diol moieties in carbohydrates and coupling to hydrazine beads [190].

Fig. 22. Strategy for the affinity enrichment of nitrotyrosine-containing peptides [193]. For details, see text.

Fig. 23. N-terminal charge derivatization reagents for the modification of peptide fragmentation.

Fig. 24. Modification of the arginine side-chain by the butanedione–phenylboronic acid tag [215].

Applications of chemical tagging strategies for the study of protein phosphorylation

References are given in alphabetical order.

Some decision-making points for the choice of chemical tagging strategies