Identification of an artifact in the mass spectrometry of proteins derivatized with iodoacetamide

Derivatization of cysteinyl residues is often used to prevent the formation of disulfide bonds during protein isolation and analysis. The most commonly used reagents are iodoacetic acid and iodoacetamide, which increase the molecular mass of the protein by 58 or 57 Da, respectively, for each derivatized cysteine. A possible side reaction is derivatization of methionine. In our analysis of derivatized human lens alphaA-crystallins, we found an apparent molecular mass 48 Da lower than the mass expected for alphaA-crystallin with the cysteines carboxyamidomethylated. Analysis of a tryptic digest of this protein showed that both cysteines and one methionine had been derivatized. Peaks indicating a molecular mass 48 Da less than expected for the protein with only cysteines derivatized were attributed to fragmentation of the derivatized methionine through collision-induced dissociation in the electrospray ionization source. An awareness of this artifact is important to investigators searching for proteins and their modified forms in complex mixtures.     

Mass spectrometric identification of formaldehyde-induced peptide modifications under in vivo protein cross-linking conditions

Formaldehyde cross-linking of proteins is emerging as a novel approach to study protein-protein interactions in living cells. It has been shown to be compatible with standard techniques used in functional proteomics such as affinity-based protein enrichment, enzymatic digestion, and mass spectrometric protein identification. So far, the lack of knowledge on formaldehyde-induced protein modifications and suitable mass spectrometric methods for their targeted detection has impeded the identification of the different types of cross-linked peptides in these samples. In particular, it has remained unclear whether in vitro studies that identified a multitude of amino acid residues reacting with formaldehyde over the course of several days are suitable substitutes for the much shorter reaction times of 10-20 min used in cross-linking experiments in living cells. The current study on model peptides identifies amino-termini as well as lysine, tryptophan, and cysteine side chains, i.e. a small subset of those modified after several days, as the major reactive sites under such conditions, and suggests relative position in the peptide sequence as well as sequence microenvironment to be important factors that govern reactivity. Using MALDI-MS, mass increases of 12 Da on amino groups and 30 Da on cysteines were detected as the major reaction products, while peptide fragment ion analysis by tandem mass spectrometry was used to localize the actual modification sites on a peptide. Non-specific cross-linking was absent, and could only be detected with low yield at elevated peptide concentrations. The detailed knowledge on the constraints and products of the formaldehyde reaction with peptides after short incubation times presented in this study is expected to facilitate the targeted mass spectrometric analysis of proteins after in vivo formaldehyde cross-linking.     

Tunable heteroaromatic azoline thioethers (HATs) for cysteine profiling

Here we report a new series of hydrolytically stable chemotype heteroaromatic azoline thioethers (HATs) to achieve highly selective, rapid, and efficient covalent labeling of cysteine under physiological conditions. Although the resulting cysteine–azoline conjugate is stable, we highlight traceless decoupling of the conjugate to afford unmodified starting components in response to reducing conditions. We demonstrated that HAT probes reverse the reactivity of nucleophilic cysteine to electrophilic dehydroalanine (Dha) under mild basic conditions. We demonstrated the umpolung capability of HAT probes for the modification of cysteine on peptides and proteins with various nucleophiles. We demonstrated that HAT probes increase the mass sensitivity of the modified peptides and proteins by 100 fold as compared to the classical methods. Finally, we extended the application of HAT probes for specific modification of cysteines in a complex cell lysate mixture.

Here we report a new series of hydrolytically stable chemotype heteroaromatic azoline thioethers (HATs) to achieve highly selective, rapid, and efficient covalent labeling of cysteine under physiological conditions.     

Mass spectrometric identification of in vivo carbamylation of the amino terminus of Ectothiorhodospira mobilis high-potential iron-sulfur protein,isozyme 1

The complete amino acid sequence of a novel high-potential iron-sulfur protein (HiPIP) isozyme 1 from the moderately halophilic phototrophic bacterium Ectothiorhodospira mobilis was determined by a combined approach of chemical and mass spectrometric sequencing techniques. By mass analysis of the apo- and holo-protein in the positive electrospray ionization mode using different electrospray solvents, the protein was found to be post-translationally modified by a moiety of 43 Da. Further analysis showed the nature and location of this modification to be a carbamyl group at the N-terminus of the HiPIP. This rare type of modification has previously been reported to occur in the water-soluble human lens alphaB-crystallin, class D beta-lactamases and some prokaryotic ureases, albeit at an internal lysine residue. In this paper, we discuss the mass spectrometric features of a carbamylated residue at the N-terminus of a peptide or a lysine side-chain during sequence analysis by collision-induced dissociation tandem mass spectrometry. Our data provide evidence for the first case of a prokaryotic carbamylated electron transport protein occurring in vivo.     

N-terminal Cyclization of Peptides in Large-scale Protein Identification Based on Biological Mass Spectrometry

Biological mass spectrometry has been developed for the large-scale protein identification. The successful identification of protein in proteomic study is based on an effective match of MS data to the sequence in database. At times, because of the diversity and heterogeneity of protein modification, the experimental data obtained by mass spectrometry does not match the theoretical value; hence, approximately 90 percent or more of the tandem mass spectra cannot be identified effectively. This has become one of the most important technique issues to be resolved in current proteome research. The N-terminal cyclization of peptides, as one of a variety of modification introduced in sample preparation, has been preliminarily studied in this work. The result showed that N-terminal cyclization occurred in most of the glutamine (Q) or carbamoylmethyl-cysteine (CAM_C) residues, and the reaction is often incomplete or partial; both types of peptides can often exist in its respective state at the same time, and the behavior of modified peptides in reversion phase chromatography is changed. The success rate of protein identification could be obviously improved by the addition of the N-terminal cyclization modification in the database searching. These results will be very helpful in the mass spectrometric data analysis of proteomic study.     

Identification and characterization of cysteinyl exposure in proteins by selective mercury labeling and nano‐electrospray ionization quadrupole time‐of‐flight mass spectrometry

We describe a method for probing surface‐exposed cysteines in proteins by selective labeling with p‐hydroxymercuribenzoate (PMB) combined with nano‐electrospray ionization mass spectrometric analysis (nanoESI‐MS). The rapid, stoichiometric, and specific labeling by PMB of surface‐exposed cysteines allows for characterization of the accessibility of the cysteines using a single MS analysis. Moreover, by taking advantage of the large mass shift of 321 Da, unique isotopic pattern, and enhanced MS signal of PMB‐labeled cysteine‐containing peptide fragments, the surface‐exposed cysteines in proteins can be accurately identified by peptide mapping. The number and sites of reactive cysteines on the surface of human and rat hemoglobins (hHb and rHb) were identified as examples. Collision‐induced dissociation tandem mass spectrometric (MS/MS) analysis of specific peptides further confirmed the selective labeling of PMB in hHb. The subtle difference between the different cysteine residues in rHb was also evaluated by multiple PMB titrations. The difference between the two cysteines in their environment may partially explain their reaction specificity. Cysteine 125 in the β unit of rHb is exposed on the surface, explaining its reactivity with glutathione. Cysteine 13 in the α subunit of rHb is much less exposed, and is located in a hydrophobic pocket, a conclusion that is consistent with the previous observation of its selective binding with dimethylarsinous acid, a reactive arsenic metabolite. The method is potentially useful for probing cysteines in other biologically important proteins and for studying proteins that are associated with conformational or structural changes induced by denaturing processes, protein modifications, protein‐protein interactions and protein assemblies. Copyright © 2010 John Wiley & Sons, Ltd.     

Mass spectrometric investigation of protein alkylation by the RNA footprinting probe kethoxal

The reactivity of the RNA footprinting reagent kethoxal (KT) toward proteins was investigated by electrospray ionization-Fourier transform mass spectrometry. Using standard peptides, KT was shown to selectively modify the guanidino group of arginine side chains at neutral pH, while primary amino groups of lysine and N-terminus were found to be unreactive under these conditions. Gas-phase fragmentation of KT adducts provided evidence for a cyclic 1,2-diol structure. Esterification of the 1,2-diol product was obtained in borate buffer, and its structure was also investigated by tandem mass spectrometry. When model proteins were probed with this RNA footprinting reagent, the adducts proved to be sufficiently stable to allow for the application of different peptide-mapping procedures to identify the location of modified arginines. Probing of proteins under native folding conditions provided modification patterns that very closely matched the structural context of arginines in the global protein structure. A strong correlation was demonstrated between the susceptibility to modification and residue accessibility calculated from the known 3D structure. When the complexes formed by HIV-1 nucleocapsid (NC) protein and RNA stemloops SL2 and SL3 were investigated, KT footprinting provided accurate information regarding the involvement of individual arginines in binding RNA and showed different reactivity according to their mode of interaction.     

Thermal stability of hybrid materials based on epoxy functional (poly)siloxanes

Epoxy functional (poly)siloxanes are one of the most important classes of modified silicones. Due to high reactivity of epoxy group and specific features of siloxane chain, they can make an excellent raw material for synthesis of hybrid materials. Results obtained in this study have shown that both the modification of epoxy resins with epoxy functional disiloxanes as well as the application of polysiloxanes with long polysiloxane chains and a specified content of epoxy groups makes it possible to produce hybrid materials of very good thermal stability. Crosslinking reactions were carried out with use of four diamines of which the best one appeared to be 4,4??-diaminodiphenylmethane. The highest thermal stability was found in the case of hybrid materials obtained from epoxy functional polysiloxanes.     

Structure-reactivity relationships among metallothionein three-metal domains: role of non-cysteine amino acid residues in lobster metallothionein and human metallothionein-3

Metallothionein (MT) domains of different origins, exhibiting distinct, highly conserved cysteine positions, show differences in metal-cysteine coordination and reactivity. Lobster MT, which includes two Cd3S9 beta domains, was chosen as a basic model to study the structure-function relationship among the clusters. The possible influence of (1) the position of the cysteine residues and (2) the steric and electrostatic effects of neighboring amino acids on the folding and stability of MT clusters have been examined with the native lobster beta C and beta N domains, each having nine cysteines and binding three M2+ ions, and a modified domain beta C-->N, in which the cysteines of the C-terminal domain are relocated so they are spaced as in the N-terminal domain. Each has been synthesized and characterized by UV, CD, 113Cd NMR, and 1H NMR spectroscopies. The synthetic native domains (Cd3 beta C and Cd3 beta N) displayed spectroscopic properties, metal-binding affinities, and kinetic reactivity similar to those of the holo protein. In contrast, the modified Cd3 beta C-->N domain was unusually reactive and, in the presence of Chelex, a metal-ion chelating resin, was converted to a Cd5(beta C-->N)2 dimer. These differences in structure and reactivity demonstrate that the requirements for formation of a stable type-B, Cd3S9, beta cluster are more stringent than simply the sequential positions of the cysteines along the peptide chain and include specific interactions with neighboring amino acids. Molecular mechanics calculations suggest that changes of even a single amino acid in lobster Cd3 beta N toward lobster Cd3 beta C-->N or in mammalian MT1 or MT2 toward Cd3 beta-MT3 (GIF) can destabilize their structures.     

Electrospray-assisted modification of proteins: a radical probe of protein structure

A new approach is described to probe the structure of proteins through their reactivity with oxygen-containing radicals. Radical-induced oxidative modification of proteins is achieved within an electrospray ion source using oxygen as a reactive nebulizer gas at high needle voltages. This method facilitates the rapid oxidation of proteins as the molecules emerge from the electrospray needle tip. Electrospray mass spectra of both ubiquitin and lysozyme reveal that over 50% of the protein can be modified under these conditions. The radical-induced oxidative modification of amino acid side chains is correlated with their solvent accessibility to obtain information on a protein's higher-order structure. The oxidation sites in hen lysozyme have been identified by proteolysis of the condensed protein solution and tandem mass spectrometry (MS/MS). Oxidation of tryptophan at positions 62 and 123 occurs exclusively over all other tryptophan residues, consistent with the relative solvent accessibilities of the residue side chains based on the NMR structure of the protein. Radical-induced oxidative modification of cysteine (Cys), methionine (Met), tryptophan (Trp), phenylalanine (Phe), tyrosine (Tyr), proline (Pro), histidine (His), and leucine (Leu) residues is also reported, providing sufficient reactive markers to span a protein sequence. This facile oxidation process could be applied to investigate the molecular mechanism by which reactive oxygen species interact with a particular protein domain as a means to investigate the onset of certain diseases.     

Multiple neutral loss monitoring (MNM): A multiplexed method for post-translational modification screening

Post-translational modifications of proteins are involved in determining the activity of proteins and are essential for proper protein function. Current mass spectrometric strategies require one to specify a particular type of modification, in some cases also a particular charge state of a protein or peptide that is to be studied before the actual analysis. Due to these requirements, most of the modifications on proteins are not considered in such an experiment and, thus, a series of similar analyses need to be performed to ensure a more extensive characterization. A novel scan strategy has been developed, multiple neutral loss monitoring (MNM), allowing for the comprehensive screening of post-translational modifications (PTM) on proteins that fragment as neutral losses in a mass spectrometer. MNM method parameters were determined by performing product ion scans on a number of modified peptides over a range of collision energies, providing neutral loss energy profiles and optimal collision energies (OCE) for each modification, supplying valuable information pertaining to the fragmentation of these modifications and the necessary parameters that would be required to obtain the best analysis. As the optimal collision energy was highly dependent on the type of modification and the charge state of the peptide, the MNM scan was operated with a collision energy gradient. Autocorrelation analyses identified the type of modification, and convolution mapping analyses identified the associated peptide. The MNM scan with the new collision energy parameters was successfully applied to a mixture of four modified peptides in a BSA digest. The implementation of this technique will allow for comprehensive screening of all modifications that fragment as neutral losses.     

Effects of As(III) binding on beta-hairpin structure

While arsenic(III) compounds can exert profound toxicological and pharmacological effects, their modes of action and, in particular, the structural consequences of their binding to cysteinyl side chains in proteins, remain poorly understood. To gain an understanding of how arsenic binding influences beta-structure, pairs of cysteines were introduced into a model monomeric beta-hairpin to yield a family of peptides such that coordination occurs either across the strands or within the same strand of the beta-hairpin. Circular dichroism, NMR, UV-vis spectroscopy, and rapid-reaction studies were used to characterize the binding of monomethylarsonous acid or p-succinylamidephenyl arsenoxide (PSAO) to these peptides. Placement of cysteines at non-hydrogen bond (NHB) positions across the beta-hairpin, such that they occupy the same face of the sheet, was found to enhance the structure as assessed by CD. Cross-strand cysteine residues that project on opposite faces close to the termini of the hairpin can still bind arsenic tightly and show modestly increased beta-sheet content. NMR and modeling studies suggest that arsenic can be accommodated at this locus without disrupting the core interactions stabilizing the turn. However, As(III) binding to nonopposed cysteines, or to cysteines at HB and NHB positions along one strand of the hairpin, caused loss of structure. UV-vis titrations show that all these hairpin peptides bind PSAO stoichiometrically with K(d) values from 13 to 106 nM. Further, binding is moderately rapid, with second-order rate constants for association of 10,000-22,000 M(-1) s(-)1 irrespective of the placement of the cysteines within the hairpin and the consequent extent of structural reorganization required as a result of binding. These studies complement recent work with alpha-helices and further demonstrate that capture of a pair of thiols by As(III) may result in significant changes in local secondary structure in the protein targets of these potent bioactive agents.     

Fast and Selective Modification of Thiol Proteins/Peptides by <Emphasis Type="Italic">N</Emphasis>-(Phenylseleno)phthalimide

We previously reported that selenamide reagents such as ebselen and N-(phenylseleno)phthalimide (NPSP) can be used to selectively derivatize thiols for mass spectrometric analysis, and the introduced selenium tags are useful as they could survive or removed with collision-induced dissociation (CID). Described herein is the further study of the reactivity of various protein/peptide thiols toward NPSP and its application to derivatize thiol peptides in protein digests. With a modified protocol (i.e., dissolving NPSP in acetonitrile instead of aqueous solvent), we found that quantitative conversion of thiols can be obtained in seconds, using NPSP in a slight excess amount (NPSP:thiol of 1.1–2:1). Further investigation shows that the thiol reactivity toward NPSP reflects its chemical environment and accessibility in proteins/peptides. For instance, adjacent basic amino acid residues increase the thiol reactivity, probably because they could stabilize the thiolate form to facilitate the nucleophilic attack of thiol on NPSP. In the case of creatine phosphokinase, the native protein predominately has one thiol reacted with NPSP while all of four thiol groups of the denatured protein can be derivatized, in accordance with the corresponding protein conformation. In addition, thiol peptides in protein/peptide enzymatic digests can be quickly and effectively tagged by NPSP following tri-n-butylphosphine (TBP) reduction. Notably, all three thiols of the peptide QCCASVCSL in the insulin peptic digest can be modified simultaneously by NPSP. These results suggest a novel and selective method for protecting thiols in the bottom-up approach for protein structure analysis.     

Selective nucleophilic substitution reactions on poly(epichlorohydrin) using aromatic and aliphatic thiol compounds

Poly(epichlorohydrin) has been modified chemically using aromatic and aliphatic thiol compounds. The reactivity and kinetics of these modifiers with respect to substitution and elimination was studied. Therefore, the chemical structure of the reaction products was analysed using ¹³C NMR, ¹H NMR and ¹³C-DEPT spectroscopies. It is shown that both, aromatic as well as aliphatic thiols, are highly selective with respect to nucleophilic substitution as reaction conditions can be found which allow one to achieve degrees of modification of up to 90% without any elimination side-reaction. As a consequence no degradative chain-scission takes place what has been confirmed by GPC analysis.A comparison between both types of thiol modifiers shows that aromatic ones react faster and that higher degrees of modification are reached than with their aliphatic homologues.     

Use of matrix-assisted laser desorption/ionization time-of-flight mass mapping and nanospray liquid chromatography/electrospray ionization tandem mass spectrometry sequence tag analysis for high sensitivity identification of yeast proteins separated by two-dimensional gel electrophoresis

Current analytical techniques in protein identification by mass spectrometry are based on the generation of peptide mass maps or sequence tags that are idiotypic for the protein sequence. This work reports on the development of the use of mass spectrometric methods for protein identification in research on metabolic pathways of a genetically modified strain of the baker's yeast Saccharomyces cerevisiae. This study describes the use of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass mapping and liquid chromatography/quadrupole time-of-flight electrospray ionization tandem mass spectrometry (LC/Q-TOF-ESI-MS/MS) sequence tag analysis in identification of yeast proteins separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The spots were selected for analysis in order to collect information for future studies, to cover the whole pI range from 3 to 10, and to evaluate information from spots of different intensities. Mass mapping as a rapid, high-throughput method was in most cases sensitive enough for identification. LC/MS/MS was found to be more sensitive and to provide more accurate data, and was very useful when analyzing small amounts of sample. Even one sequence tag acquired by this method could be enough for unambiguous identification, and, in the present case, successfully identified a point mutation.     

Accelerated on-column lysine derivatization and cysteine methylation by imidazole reaction in a deuterated environment for enhanced product ion analysis

The combination of separation techniques and mass spectrometry (MS) for peptide investigation allows superior sensitivity of detection and richer fragmentation data than available by direct MS analysis of a complex mixture. In this regard, liquid chromatography (LC) coupled to electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) MS have evolved as versatile analytical tools in proteomics. Very often, however, the product ion mass spectrum is either incomplete or overfilled with ions, thus making sequence analysis difficult. Here we report overall ion intensity improvement of C-terminal lysine-containing peptides from Lys-C digest by on-column derivatization of lysines with 2-methoxy-4,5-dihydro-1H-imidazole. The method is simple, fast and exhibits 100% efficiency of the reaction. Additionally, post-source decay carried out on derivatized peptides gave rise almost exclusively to y-series ion formation, at 100% sequence coverage and high intensity. The novelty of the method resides in the side reaction of this derivatization process, namely the methylation of cysteines. This facilitates the estimation of the disulfide bridge position in a protein and the fragmentation of cysteine-containing peptide fragments. Additionally, by using this derivatization procedure, the loss of peptides, their degradation and/or oxidation, usually occurring in digest alkylation procedures, is greatly minimized. The new on-column derivatization protocol is designed to be carried out on C18 Spin Tubes or Cleanup C18 Pipette Tips. We observed that use of buffered D2O solvent prevented unwanted oxidation and degradation reactions with respect to the stationary phase. This may be due to the fact that a deuteron is less polar than a proton, and thus the bonded silica stationary phase saturated with deuterons does not affect the reaction between epsilon-amino or cysteine thiol groups and 2-methoxy-4,5-dihydro-1H-imidazole. Complete tagging of the peptides by on-column reaction could be obtained when using D2O, as compared to water-based reaction. Methylation of cysteine residues was enhanced when beta-mercaptoethanol was added in the reactant solution.     

Covalent modification of actin by 4-hydroxy-trans-2-nonenal (HNE): LC-ESI-MS/MS evidence for Cys374 Michael adduction

We demonstrate for the first time, by a combined mass spectrometric and computational approach, that G- and F-actin can be covalently modified by the lipid-derived aldehyde, 4-hydroxy-trans-2-nonenal, providing information on the molecular mass of modified protein and the mechanism and site of adduction.ESI-MS analysis of actin treated with different molar ratios of HNE (1 : 1 to 1 : 20) showed the formation of a protein derivative in which there was an increase of 156 Da (42028 Da) over native actin (41872 Da), consistent with the adduction of one HNE residue through Michael addition. To identify the site of HNE adduction, G- and F-actin were stabilized by NaBH(4) reduction and digested with trypsin. LC-ESI-MS/MS analysis in data-dependent scan mode of the resulting peptides unequivocally indicated that Cys374 is the site of HNE adduction. Computational studies showed that the reactivity of Cys374 residue is due to a significant accessible surface and substantial thiol acidity due to the particular microenvironment surrounding Cys374.     

Automated data interpretation based on the concept of "negative signature mass" for mass-mapping disulfide structures of cystinyl proteins

An efficient method for data processing and interpretation is needed to support and extend disulfide mass-mapping methodology based on partial reduction and cyanylation-induced cleavage to proteins containing more than four cystines. Here, the concept of "negative signature mass" is introduced as the novel feature of an algorithm designed to identify the disulfide structure of a cystinyl protein given an input of mass spectral data and an amino acid sequence. The "negative signature mass" process is different from the conventional approach in that it does not directly rule-in disulfide linkages, but rather eliminates linkages from a list of all possible theoretical linkages, with the goal of ruling out enough linkages so that only one disulfide structure can be constructed. The operating principles and the effectiveness of the algorithm are described in the context of analyzing ribonuclease A, a 124-residue protein containing eight cysteines in the form of four cystines (disulfides).     

Studying disulfide bond rearrangement by MALDI-RTOF PSD and MALDI-TOF/RTOF high-energy CID (20 keV) experiments of peptides derived from ammodytoxins

Ammodytoxins (Atxs) are presynaptically neurotoxic phospholipases present in Vipera ammodytes ammodytes snake venom. Atxs show a high sequence homology and contain 14 cysteines which form seven biologically relevant disulfide bridges-connecting non-neighboring cysteines. Formic acid cleavage was performed to confirm protein sequences by MALDI RTOF MS and resulted in 95.6% sequence coverage exhibiting only few formylations. Cysteine-containing peptides showed adjacent signals 2 and/or 4 Da lower (according to the number of cysteines present in the peptide) than the theoretical molecular weight indicating disulfide bridge rearrangement. Post-source decay (PSD) and high-energy collision-induced dissociation (CID) at 20 keV experiments showed fragmentation pattern unique for the reduced, thiol group containing and the oxidized, disulfide bridge harboring peptides. Besides typical low-energy fragment ions observed during PSD experiments (a-, b-, y-type ions), additional high-energy fragment ions (c-, x-, w-, d-type and internal fragments) of significant intensity were generated during fragmentation at 20 keV. In the case of charge directing N- and C-termini, x- and w-type ions were also observed during PSD. Good and up to complete sequence coverage was achieved for all studied peptides from Atxs in the case of high-energy CID, whereas PSD lacked information particularly for larger peptides.     

New Protein Footprinting: Fast Photochemical Iodination Combined with Top-Down and Bottom-Up Mass Spectrometry

We report a new approach for the fast photochemical oxidation of proteins (FPOP) whereby iodine species are used as the modifying reagent. We generate the radicals by photolysis of iodobenzoic acid at 248 nm; the putative iodine radical then rapidly modifies the target protein. This iodine-radical labeling is sensitive, tunable, and site-specific, modifying only histidine and tyrosine residues in contrast to OH radicals that modify 14 amino-acid side chains. We iodinated myoglobin (Mb) and apomyoglobin (aMb) in their native states and analyzed the outcome by both top-down and bottom-up proteomic strategies. Top-down sequencing selects a certain level (addition of one I, two I's) of modification and determines the major components produced in the modification reaction, whereas bottom-up reveals details for each modification site. Tyr146 is found to be modified for aMb but less so for Mb. His82, His93, and His97 are at least 10 times more modified for aMb than for Mb, in agreement with NMR studies. For carbonic anhydrase and its apo form, there are no significant differences of the modification extents, indicating their similarity in conformation and providing a control for this approach. For lispro insulin, insulin-EDTA, and insulin complexed with zinc, iodination yields are sensitive to differences in insulin oligomerization state. The iodine radical labeling is a promising addition to protein footprinting methods, offering higher specificity and lower reactivity than ?OH and SO(4)(-?), two other radicals already employed in FPOP.