Defining the interacting regions between apomyoglobin and lipid membrane by hydrogen/deuterium exchange coupled to mass spectrometry

Sperm whale myoglobin can be considered as the model protein of the globin family. The pH-dependence of the interactions of apomyoglobin with lipid bilayers shares some similarities with the behavior of pore-forming domains of bacterial toxins belonging also to the globin family. Two different states of apomyoglobin bound to a lipid bilayer have been characterized by using hydrogen/deuterium exchange experiments and mass spectrometry. When bound to the membrane at pH 5.5, apomyoglobin remains mostly native-like and interacts through alpha-helix A. At pH 4, the binding is related to the stabilization of a partially folded state. In that case, alpha-helices A and G are involved in the interaction. At this pH, alpha-helix G, which is the most hydrophobic region of apomyoglobin, is available for interaction with the lipid bilayer because of the loss of the tertiary structure. Our results show the feasibility of such experiments and their potential for the characterization of various membrane-bound states of amphitropic proteins such as pore-forming domains of bacterial toxins. This is not possible with other high-resolution methods, because these proteins are usually in partially folded states when interacting with membranes.     

Hydrogen/deuterium exchange mass spectrometric analysis of conformational changes accompanying the assembly of the yeast prion Ure2p into protein fibrils

The Ure2 protein from baker's yeast (Saccharomyces cerevisiae) has prion properties. In vitro, at neutral pH, soluble Ure2p forms long, twisted fibrils. Two models have been proposed to account for Ure2p polymerization. The first postulates that a segment of 70 amino acid residues in the flexible N-terminal domain from different Ure2p molecules forms a parallel superpleated beta-structure running along the fibrils. The second hypothesizes that assembly of full-length Ure2p is driven by limited conformational rearrangements and non-native inter- and intramolecular interactions. The knowledge of the three-dimensional structure of the fibrillar form of Ure2p is critical for understanding the molecular events leading to the polymerization of soluble Ure2p into fibrils and hence for the design of inhibitors that might have therapeutic potential as yeast prions possessing domains rich in N and Q residues, similar to huntingtin. Solvent-accessibility studies using hydrogen/deuterium exchange monitored by mass spectrometry (HXMS) can provide insights into the structure of the fibrillar form of Ure2p and characterize at the molecular level the conformational rearrangements that occur upon assembly, in particular through the identification of protected regions and their localization in the overall structure of the protein. We have analyzed the changes in Ure2p structure associated with its assembly into fibrils using HXMS. The deuterium incorporation profile along the sequence allows the identification of the regions that exhibit the most important conformational change. Our data reveal that Ure2p undergoes minor structural changes upon assembly. While polypeptides [82-92] and [13-37] exhibit significant increased and decreased exposure to the solvent, respectively, no marked change was observed for the rest of the protein upon assembly. Our results afford new insights into the conformational rearrangements that lead to the assembly of Ure2p into fibrils and the propagation of the [URE3] element in yeast.     

PAS domain allostery and light-induced conformational changes in photoactive yellow protein upon I2 intermediate formation, probed with enhanced hydrogen/deuterium exchange mass spectrometry

Photoactive yellow protein (PYP) is a small bacterial photoreceptor that undergoes a light-activated reaction cycle. PYP is also the prototypical Per-Arnt-Sim (PAS) domain. PAS domains, found in diverse multi-domain proteins from bacteria to humans, mediate protein-protein interactions and function as sensors and signal transducers. Here, we investigate conformational and dynamic changes in solution in wild-type PYP upon formation of the long-lived putative signaling intermediate I2 with enhanced hydrogen/deuterium exchange mass spectrometry (DXMS). The DXMS results showed that the central beta-sheet remains stable but specific external protein segments become strongly deprotected. Light-induced disruption of the dark-state hydrogen bonding network in I2 produces increased flexibility and opening of PAS core helices alpha3 and alpha4, releases the beta4-beta5 hairpin, and propagates conformational changes to the central beta-sheet. Surprisingly, the first approximately 10 N-terminal residues, which are essential for fast dark-state recovery from I2, become more protected. By combining the DXMS results with our crystallographic structures, which reveal detailed changes near the chromophore but limited protein conformational change, we propose a mechanism for I2 state formation. This mechanism integrates the results from diverse biophysical studies of PYP, and links an allosteric T to R-state conformational transition to three pathways for signal propagation within the PYP fold. On the basis of the observed changes in PYP plus commonalities shared among PAS domain proteins, we further propose that PAS domains share this conformational mechanism, which explains the versatile signal transduction properties of the structurally conserved PYP/PAS module by framework-encoded allostery.     

Scrapie prion protein structural constraints obtained by limited proteolysis and mass spectrometry

Elucidation of the structure of scrapie prion protein (PrP^Sc), essential to understand the molecular mechanism of prion transmission, continues to be one of the major challenges in prion research and is hampered by the insolubility and polymeric character of PrP^Sc. Limited proteolysis is a useful tool to obtain insight on structural features of proteins: proteolytic enzymes cleave proteins more readily at exposed sites, preferentially within loops, and rarely in β-strands. We treated PrP^Sc isolated from brains of hamsters infected with 263K and drowsy prions with varying concentrations of proteinase K (PK). After PK deactivation, PrP^Sc was denatured, reduced, and cleaved at Cys179 with 2-nitro-5-thiocyanatobenzoic acid. Fragments were analyzed by nano-HPLC/mass spectrometry and matrix-assisted laser desorption/ionization. Besides the known cleavages at positions 90, 86, and 92 for 263K prions and at positions 86, 90, 92, 98, and 101 for drowsy prions, our data clearly demonstrate the existence of additional cleavage sites at more internal positions, including 117, 119, 135, 139, 142, and 154 in both strains. PK concentration dependence analysis and limited proteolysis after partial unfolding of PrP^Sc confirmed that only the mentioned cleavage sites at the N-terminal side of the PrP^Sc are susceptible to PK. Our results indicate that besides the “classic” amino-terminal PK cleavage points, PrP^Sc contains, in its middle core, regions that show some degree of susceptibility to proteases and must therefore correspond to subdomains with some degree of structural flexibility, interspersed with stretches of amino acids of high resistance to proteases. These results are compatible with a structure consisting of short β-sheet stretches connected by loops and turns.     

Probing the self-assembly and the accompanying structural changes of hydrophobin SC3 on a hydrophobic surface by mass spectrometry

The fungal class I hydrophobin SC3 self-assembles into an amphipathic membrane at hydrophilic-hydrophobic interfaces such as the water-air and water-Teflon interface. During self-assembly, the water-soluble state of SC3 proceeds via the intermediate alpha-helical state to the stable end form called the beta-sheet state. Self-assembly of the hydrophobin at the Teflon surface is arrested in the alpha-helical state. The beta-sheet state can be induced at elevated temperature in the presence of detergent. The structural changes of SC3 were monitored by various mass spectrometry techniques. We show that the so-called second loop of SC3 (C39-S72) has a high affinity for Teflon. Binding of this part of SC3 to Teflon was accompanied by the formation of alpha-helical structure and resulted in low solvent accessibility. The solvent-protected region of the second loop extended upon conversion to the beta-sheet state. In contrast, the C-terminal part of SC3 became more exposed to the solvent. The results indicate that the second loop of class I hydrophobins plays a pivotal role in self-assembly at the hydrophilic-hydrophobic interface. Of interest, this loop is much smaller in case of class II hydrophobins, which may explain the differences in their assembly.     

Identification of fungal microorganisms by MALDI-TOF mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as a reliable tool for fast identification and classification of microorganisms. In this regard, it represents a strong challenge to microscopic and molecular biology methods. Nowadays, commercial MALDI systems are accessible for biological research work as well as for diagnostic applications in clinical medicine, biotechnology and industry. They are employed namely in bacterial biotyping but numerous experimental strategies have also been developed for the analysis of fungi, which is the topic of the present review. Members of many fungal genera such as Aspergillus, Fusarium, Penicillium or Trichoderma and also various yeasts from clinical samples (e.g. Candida albicans) have been successfully identified by MALDI-TOF MS. However, there is no versatile method for fungi currently available even though the use of only a limited number of matrix compounds has been reported. Either intact cell/spore MALDI-TOF MS is chosen or an extraction of surface proteins is performed and then the resulting extract is measured. Biotrophic fungal phytopathogens can be identified via a direct acquisition of MALDI-TOF mass spectra e.g. from infected plant organs contaminated by fungal spores. Mass spectrometric peptide/protein profiles of fungi display peaks in the m/z region of 1000–20 000, where a unique set of biomarker ions may appear facilitating a differentiation of samples at the level of genus, species or strain. This is done with the help of a processing software and spectral database of reference strains, which should preferably be constructed under the same standardized experimental conditions.     

Analysis of N-glycosylation in maize cytokinin oxidase/dehydrogenase 1 using a manual microgradient chromatographic separation coupled offline to MALDI-TOF/TOF mass spectrometry

Cytokinin oxidase/dehydrogenase (CKO; EC 1.5.99.12) irreversibly degrades the plant hormones cytokinins. A recombinant maize isoenzyme 1 (ZmCKO1) produced in the yeast Yarrowia lipolytica was subjected to enzymatic deglycosylation by endoglycosidase H. Spectrophotometric assays showed that both activity and thermostability of the enzyme decreased after the treatment at non-denaturing conditions indicating the biological importance of ZmCKO1 glycosylation. The released N-glycans were purified with graphitized carbon sorbent and analyzed by MALDI-TOF MS. The structure of the measured high-mannose type N-glycans was confirmed by tandem mass spectrometry (MS/MS) on a Q-TOF instrument with electrospray ionization. Further experiments were focused on direct analysis of sugar binding. Peptides and glycopeptides purified from tryptic digests of recombinant ZmCKO1 were separated by reversed-phase chromatography using a manual microgradient device; the latter were then subjected to offline-coupled analysis on a MALDI-TOF/TOF instrument. Glycopeptide sequencing by MALDI-TOF/TOF MS/MS demonstrated N-glycosylation at Asn52, 63, 134, 294, 323 and 338. The bound glycans contained 3-14 mannose residues. Interestingly, Asn134 was found only partially glycosylated. Asn338 was the sole site to carry large glycan chains exceeding 25 mannose residues. This observation demonstrates that contrary to a previous belief, the heterologous expression in Y. lipolytica may lead to locally hyperglycosylated proteins.     

Mapping the structure of folding cores in TIM barrel proteins by hydrogen exchange mass spectrometry: the roles of motif and sequence for the indole-3-glycerol phosphate synthase from Sulfolobus solfataricus

To test the roles of motif and amino acid sequence in the folding mechanisms of TIM barrel proteins, hydrogen-deuterium exchange was used to explore the structure of the stable folding intermediates for the of indole-3-glycerol phosphate synthase from Sulfolobus solfataricus (sIGPS). Previous studies of the urea denaturation of sIGPS revealed the presence of an intermediate that is highly populated at approximately 4.5 M urea and contains approximately 50% of the secondary structure of the native (N) state. Kinetic studies showed that this apparent equilibrium intermediate is actually comprised of two thermodynamically distinct species, I(a) and I(b). To probe the location of the secondary structure in this pair of stable on-pathway intermediates, the equilibrium unfolding process of sIGPS was monitored by hydrogen-deuterium exchange mass spectrometry. The intact protein and pepsin-digested fragments were studied at various concentrations of urea by electrospray and matrix-assisted laser desorption ionization time-of-flight mass spectrometry, respectively. Intact sIGPS strongly protects at least 54 amide protons from hydrogen-deuterium exchange in the intermediate states, demonstrating the presence of stable folded cores. When the protection patterns and the exchange mechanisms for the peptides are considered with the proposed folding mechanism, the results can be interpreted to define the structural boundaries of I(a) and I(b). Comparison of these results with previous hydrogen-deuterium exchange studies on another TIM barrel protein of low sequence identify, alpha-tryptophan synthase (alphaTS), indicates that the thermodynamic states corresponding to the folding intermediates are better conserved than their structures. Although the TIM barrel motif appears to define the basic features of the folding free energy surface, the structures of the partially folded states that appear during the folding reaction depend on the amino acid sequence. Markedly, the good correlation between the hydrogen-deuterium exchange patterns of sIGPS and alphaTS with the locations of hydrophobic clusters defined by isoleucine, leucine, and valine residues suggests that branch aliphatic side-chains play a critical role in defining the structures of the equilibrium intermediates.     

Proteome of the Escherichia coli envelope and technological challenges in membrane proteome analysis

The envelope of Escherichia coli is a complex organelle composed of the outer membrane, periplasm-peptidoglycan layer and cytoplasmic membrane. Each compartment has a unique complement of proteins, the proteome. Determining the proteome of the envelope is essential for developing an in silico bacterial model, for determining cellular responses to environmental alterations, for determining the function of proteins encoded by genes of unknown function and for development and testing of new experimental technologies such as mass spectrometric methods for identifying and quantifying hydrophobic proteins. The availability of complete genomic information has led several groups to develop computer algorithms to predict the proteome of each part of the envelope by searching the genome for leader sequences, β-sheet motifs and stretches of α-helical hydrophobic amino acids. In addition, published experimental data has been mined directly and by machine learning approaches. In this review we examine the somewhat confusing available literature and relate published experimental data to the most recent gene annotation of E. coli to describe the predicted and experimental proteome of each compartment. The problem of characterizing integral versus membrane-associated proteins is discussed. The E. coli envelope proteome provides an excellent test bed for developing mass spectrometric techniques for identifying hydrophobic proteins that have generally been refractory to analysis. We describe the gel based and solution based proteome analysis approaches along with protein cleavage and proteolysis methods that investigators are taking to tackle this difficult problem.     

R-subunit isoform specificity in protein kinase A: distinct features of protein interfaces in PKA types I and II by amide H/2H exchange mass spectrometry

The two isoforms (RI and RII) of the regulatory (R) subunit of cAMP-dependent protein kinase or protein kinase A (PKA) are similar in sequence yet have different biochemical properties and physiological functions. To further understand the molecular basis for R-isoform-specificity, the interactions of the RIIβ isoform with the PKA catalytic (C) subunit were analyzed by amide H/²H exchange mass spectrometry to compare solvent accessibility of RIIβ and the C subunit in their free and complexed states. Direct mapping of the RIIβ-C interface revealed important differences between the intersubunit interfaces in the type I and type II holoenzyme complexes. These differences are seen in both the R-subunits as well as the C-subunit. Unlike the type I isoform, the type II isoform complexes require both cAMP-binding domains, and ATP is not obligatory for high affinity interactions with the C-subunit. Surprisingly, the C-subunit mediates distinct, overlapping surfaces of interaction with the two R-isoforms despite a strong homology in sequence and similarity in domain organization. Identification of a remote allosteric site on the C-subunit that is essential for interactions with RII, but not RI subunits, further highlights the considerable diversity in interfaces found in higher order protein complexes mediated by the C-subunit of PKA.     

Echinococcus multilocularis: Proteomic analysis of the protoscoleces by two-dimensional electrophoresis and mass spectrometry

Echinococcus multilocularis is an important parasite that causes human alveolar echinococcosis. Identification and characterization of the proteins encoded by E. multilocularis metacestode might help to understand the complexity of the parasites and their interactions with the host, and to identify new candidates for immunodiagnosis and vaccine development. Here we present a proteomic analysis of E. multilocularis protoscolex (PSC) proteins. The proteins were resolved by 2-DE (pH range 3.5-10), followed by MALDI-TOF MS analysis. Fourteen known Echinococcus proteins were identified, including cytoskeletal proteins, heat shock proteins, metabolic enzymes, 14-3-3 protein, antigen P-29 and calreticulin. To construct a systematic reference map of the immunogenic proteins from E. multilocularis PSC, immunoblot analysis of PSC 2-DE maps was performed. Over 50 proteins spots were detected on immunoblots as antigens and 15 of them were defined. The results showed that cytoskeletal proteins and heat shock proteins were immunodominant antigens in alveolar echinococcosis.     

Imaging mass spectrometry reveals characteristic changes in triglyceride and phospholipid species in regenerating mouse liver

After partial hepatectomy (PH), regenerating liver accumulates unknown lipid species. Here, we analyzed lipids in murine liver and adipose tissues following PH by thin-layer chromatography (TLC), imaging mass spectrometry (IMS), and real-time RT-PCR. In liver, IMS revealed that a single TLC band comprised major 19 TG species. Similarly, IMS showed a single phospholipid TLC band to be major 13 species. In adipose tissues, PH induced changes to expression of genes regulating lipid metabolism. Finally, IMS of phosphatidylcholine species demonstrated distribution gradients in lobules that resembled hepatic zonation. IMS is thus a novel and power tool for analyzing lipid species with high resolution.     

Impact of pro segments on the folding and function of human neutrophil alpha-defensins

Human neutrophil alpha-defensins (HNPs) are synthesized in vivo as inactive precursor proteins, i.e. preproHNPs. A series of sequential proteolytic events excise the N-terminal inhibitory pro peptide, leading to defensin maturation and storage in azurophilic granules. The anionic pro peptide, required for correct sub-cellular trafficking and sorting of proHNPs, inhibits the antimicrobial activity of cationic defensins, either inter or intra-molecularly, presumably through charge neutralization. To better understand the role of the pro peptide in the folding and functioning of alpha-defensins and/or pro alpha-defensins, we chemically attached the proHNP1 pro peptide or (wt)pro peptide and the following artificial pro segments to the N terminus of HNP1: polyethylene glycol (PEG), Arg(10) (polyR), Ser(10) (polyS), and (cr)pro peptide, a charge-reversing mutant of the pro peptide where Arg/Lys residues were changed to Asp, and Asp/Glu residues to Lys. Comparative in vitro folding suggested that while all artificial pro segments chaperoned defensin folding, with PEG being the most efficient, the pro peptide catalyzed the folding of proHNPs likely through two independent mechanisms: solubilization of and interaction with the C-terminal defensin domain. Further, the N-terminal artificial pro segments dramatically altered the bactericidal activity of HNP1 against both Escherichia coli and Staphylococcus aureus. Surprisingly, (cr)pro peptide and (wt)pro peptide showed similar properties with respect to intra-molecular and inter-molecular catalysis of defensin folding as well as alpha-defensin binding, although their binding modes appeared different. Our findings identify a dual chaperone activity of the pro peptide and may shed light on the molecular mechanisms by which pro alpha-defensins fold in vivo.     

Using chemical derivatization and mass spectrometric analysis to characterize the post-translationally modified Staphylococcus aureus surface protein G

The Staphylococcus aureus surface protein G (SasG) is an important mediator of biofilm formation in virulent S. aureus strains. A detailed analysis of its primary sequence has not been reported to date. SasG is highly abundant in the cell wall of the vancomycin-intermediate S. aureus strain HIP5827, and was purified and subjected to sequence analysis by MS. Data from MALDI-TOF and LC-MS/MS experiments confirmed the predicted N-terminal signal peptide cleavage site at residue A₅₁ and the C-terminal cell wall anchor site at residue T₁₀₈₆. The protein was also derivatized with N-succinimidyloxycarbonyl-methyl-tris(2,4,6-trimethoxyphenyl) phosphonium bromide (TMPP-Ac-OSu) to assess the presence of additional N-terminal sites of mature SasG. TMPP-derivatized SasG peptides featured m/z peaks with a 572 Da mass increase over the equivalent underivatized peptides. Multiple N-terminal peptides, all of which were observed in the 150 amino acid segment following the signal peptide cleavage at the residue A₅₁, were characterized from MS and MS/MS data, suggesting a series of successive N-terminal truncations of SasG. A strategy combining TMPP derivatization, multiple enzyme digestions to generate overlapping peptides and detailed MS analysis will be useful to determine and understand functional implications of PTMs in bacterial cell wall-anchored proteins, which are frequently involved in the modulation of virulence-associated bacterial surface properties.     

Dipeptidyl peptidase 9 (DPP9) from bovine testes: Identification and characterization as the short form by mass spectrometry

The dipeptidyl peptidases (DPP) 8 and 9 belong to the DPP4 activity and/or structure homologues (DASH). Recently, a DPP9-like protein was purified from bovine testes. The aim of the present study was to prove its identity and to investigate the characteristics of this natural enzyme. We report the identification and N-terminal sequence analysis by MALDI-TOF/TOF MS, of the purified bovine enzyme as DPP9. The tryptic peptides after in-gel digestion covered 41% and 38% of the short and full-length variants of bovine DPP9, respectively. Using Asp-N digestion combined with a very recently described mass spectrometric method using DITC glass beads, the N-terminal peptide (XTGALTSERG) was isolated. It corresponds to the N-terminus of the short form of bovine DPP9. There was no evidence for glycosylation of purified bovine DPP9. The purified DPP9 was activated and stabilized by DTT. Bovine DPP9 lost its activity almost completely after alkylation with N-ethylmaleimide. Also alkylation with iodoacetamide inhibited DPP9, albeit only 70%. Other properties of bovine DPP9 are reported, including functional stability and sensitivity towards metal ions. Our results indicate that the short form of DPP9 can be isolated from bovine testes and that it behaves as a stable enzyme suitable for further functional and biochemical characterization as well as for inhibitor screening and characterization.     

Probing the dynamics of intact cells and nuclear envelope precursor membrane vesicles by deuterium solid state NMR spectroscopy

Membrane dynamics is an essential part of many cellular mechanisms such as intracellular trafficking, membrane fusion/fission and mitotic organelle reconstitution. The dynamics of membranes is dependent primarily on their phospholipid and cholesterol composition and how these molecules are ordered in relation to one another. To determine the physical status of membranes in whole cells or purified membranes of subcellular compartments we have developed a novel application exploiting solid-state ²H-NMR spectroscopy. We utilise this method to probe the dynamics of intact sperm and nuclear envelope precursor membranes. We show, using mass spectrometry, that either multilamellar or small unilamellar vesicles of deuterium-labelled palmitoyl-oleoylphosphatidylcholine can be used to probe the dynamics of sperm cells or nuclear envelope precursor membrane vesicles, respectively. Using ²H-NMR we determine the order parameters of sperm cells and nuclear envelope precursor membrane vesicles. We demonstrate that whole sperm membranes are more dynamic than nuclear envelope precursor membranes due to the higher cholesterol levels of the latter. Our new application can be exploited as a generic method for monitoring membrane dynamics in whole cells, various subcellular membrane compartments and membrane domains in subcellular compartments.     

Bacillusanthracis Surface-Layer Proteins Assemble by Binding to the Secondary Cell Wall Polysaccharide in a Manner that Requires csaB and tagO

Bacillus anthracis, the causative agent of anthrax, requires surface (S)-layer proteins for the pathogenesis of infection. Previous work characterized S-layer protein binding via the surface layer homology domain to a pyruvylated carbohydrate in the envelope of vegetative forms. The molecular identity of this carbohydrate and the mechanism of its display in the bacterial envelope are still unknown. Analyzing acid-solubilized, purified carbohydrates by mass spectrometry and NMR spectroscopy, we identify secondary cell wall polysaccharide (SCWP) as the ligand of S-layer proteins. In agreement with the model that surface layer homology domains bind to pyruvylated carbohydrate, SCWP was observed to be linked to pyruvate in a manner requiring csaB, the only structural gene known to be required for S-layer assembly. B. anthracis does not elaborate wall teichoic acids; however, its genome harbors tagO and tagA, genes responsible for the synthesis of the linkage unit that tethers teichoic acids to the peptidoglycan layer. The tagO gene appears essential for B. anthracis growth and complements the tagO mutant phenotypes of staphylococci. Tunicamycin-mediated inhibition of TagO resulted in deformed, S-layer-deficient bacilli. Together, these results suggest that tagO-mediated assembly of linkage units tethers pyruvylated SCWP to the B. anthracis envelope, thereby enabling S-layer assembly and providing for the pathogenesis of anthrax infections.     

Detection of oxidized and glycated proteins in clinical samples using mass spectrometry — A user's perspective

Background

Proteins in human tissues and body fluids continually undergo spontaneous oxidation and glycation reactions forming low levels of oxidation and glycation adduct residues. Proteolysis of oxidised and glycated proteins releases oxidised and glycated amino acids which, if they cannot be repaired, are excreted in urine.

Scope of Review

In this review we give a brief background to the classification, formation and processing of oxidised and glycated proteins in the clinical setting. We then describe the application of stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry (LC-MS/MS) for measurement of oxidative and glycation damage to proteins in clinical studies, sources of error in pre-analytic processing, corroboration with other techniques – including how this may be improved – and a systems approach to protein damage analysis for improved surety of analyte estimations.

Major conclusions

Stable isotopic dilution analysis LC-MS/MS provides a robust reference method for measurement of protein oxidation and glycation adducts. Optimised pre-analytic processing of samples and LC-MS/MS analysis procedures are required to achieve this.

General significance

Quantitative measurement of protein oxidation and glycation adducts provides information on level of exposure to potentially damaging protein modifications, protein inactivation in ageing and disease, metabolic control, protein turnover, renal function and other aspects of body function. Reliable and clinically assessable analysis is required for translation of measurement to clinical diagnostic use. Stable isotopic dilution analysis LC-MS/MS provides a “gold standard” approach and reference methodology to which other higher throughput methods such as immunoassay and indirect methods are preferably corroborated by researchers and those commercialising diagnostic kits and reagents. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

Quantification of the efficiency of cargo delivery by peptidic and pseudo-peptidic Trojan carriers using MALDI-TOF mass spectrometry

We have measured the efficiencies of two novel pseudo-peptidic carriers and various cell-penetrating peptides (Penetratin, (Arg)₉ and the third helix of the homeodomain of Knotted-1) to deliver the same cargo inside cells. The cargo that was studied corresponds to the pseudo-substrate of protein kinase C. Cargo delivery was quantified using a recent method based on isotope labeling and MALDI-TOF MS. Results of cargo delivery were compared to the amounts of free CPP internalized inside cells. The third helix of Knotted gave the best results concerning free CPP cellular uptake. It was also found to be the most efficient carrier. This peptide thus emerges as a new CPP with very promising properties.