Studies of Peptide Binding to Allyl Amine and Vinyl Acetic Acid-Modified Polymers Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (original) (raw)
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Journal of the American Society for Mass Spectrometry, 2000
Polymer surface-peptide binding interactions have been shown previously to lead to reductions in peptide matrix assisted laser desorption/ionization (MALDI) ion signals. In previous studies, increases in surface-peptide binding were characterized by the increases in both the initially adsorbed and retained quantities of 125 I-radiolabeled peptides. The present studies establish a specific correlation between the peptide retention properties of the polymer surface and the reduction in the peptide MALDI ion signal. This correlation is demonstrated by obtaining MALDI mass spectra of angiotensin I applied to various polymer surfaces having a range of peptide adsorption and retention properties. In addition, the use of a MALDI based method of standard additions is shown to allow the quantitation of the polymer surfacepeptide retention affinity for angiotensin I and porcine insulin. The MALDI standard additions method for measurement of surface-peptide retention affinities offers a number of significant advantages over conventional radiolabeled peptide binding methods and promises to be a valuable tool for the determination of this important biomaterial characteristic. (J Am Soc Mass Spectrom 2000, 11, 62-68)
Effects of Protein−Surface Interactions on Protein Ion Signals in MALDI Mass Spectrometry
Analytical Chemistry, 1999
The influence of polymer surface-protein binding affinity on protein ion signals in matrix-assisted laser desorption/ ionization (MALDI) mass spectrometry is examined. The surfaces of poly(vinylidene fluoride) and poly(ethylene terephthalate) polymer substrates are modified by pulsed rf plasma deposition of allylamine. By varying the on/off duty cycle of the pulsed rf plasma, the polymer substrate surfaces are coated with thin films having varying densities of surface amine groups. The varying surface amine density is shown to lead to systematic changes in the surface binding affinity for the 125 I-radiolabeled peptides angiotensin I and porcine insulin. Unlabeled angiotensin I and porcine insulin are then deposited on the pulsed rf plasma-modified substrates and analyzed by MALDI mass spectrometry. The experimental approach involves applying the peptide to the modified polymer surface in an aqueous phosphate-buffered saline solution and allowing the peptide solution to dry completely under ambient conditions. Subsequently, the MALDI matrix r-cyano-4hydroxycinnamic acid in methanol and 10% trifluoroacetic acid in water are added to the peptide-coated modified polymer surfaces. The results of these studies demonstrate that, for the sample preparation method employed, increases in the surface peptide binding affinity lead to decreases in the peptide MALDI ion signal.
A study of peptide–peptide interaction by matrix-assisted laser desorption/ionization
Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was used to study peptide-peptide interaction. The interaction was seen when 6-aza-2-thiothymine was used as a matrix (pH 5.4), but was disrupted with a more acidic matrix, ␣-cyano-4-hydroxycinnamic acid (pH 2.0). In the present study, we show that dynorphin, an opioid peptide, and five of its fragments that contain two adjacent basic residues (Arg 6 -Arg 7 ), all interact noncovalently with peptides that contain two to five adjacent acidic residues (Asp or Glu). Two other nonrelated peptides containing two (Arg 6 -Arg 7 ) or three (Arg 1 -Lys 2 -Arg 3 ) adjacent basic amino acid residues were studied and exhibited the same behavior. However, peptides containing adjacent Lys or His did not form noncovalent complexes with acidic peptides. The noncovalent bonding was sufficiently stable that digestion with trypsin only cleaved Arg and Lys residues that were not involved in hydrogen bonding with the acidic residues. In an equimolar mixture of dynorphin, dynorphin fragments (containing the motif RR), and an acidic peptide (minigastrin), the acidic peptide preferentially complexed with dynorphin. If the concentration of minigastrin was increased 10 fold, noncovalent interaction was seen with dynorphin and all its fragments containing the motif RR. In the absence of dynorphin, minigastrin formed noncovalent complexes with all dynorphin fragments. These findings suggest that conformation, equilibrium, and concentration do play a role in the occurrence of peptide-peptide interaction. Observations from this study include: (1) ionic bonds were not disrupted by enzymatic digests, (2) conformation and concentration influenced complex formation, and (3) the complex did not form with fragments of dynorphin or unrelated peptides that did not contain the motifs RR or RKR, nor with a fragment of dynorphin where Arg 7 was mutated to a phenylalanine residue. These findings strongly suggest that peptide-peptide interaction does occur, and can be studied by MALDI if near physiologic pH is maintained. (J Am Soc Mass Spectrom 2001, 12, 88 -96)
International Journal of Mass Spectrometry, 2018
Mass spectrometry (MS) is an essential analytical tool for the characterization of the structure of biological macromolecules, including protein-protein and protein-ligand complexes. One-dimensional MS separates gas-phase analyte ions based on their mass to charge ratio (m/z); however, to obtain more detailed structural information, tandem MS (MS/MS), which involves isolation and subsequent fragmentation of a precursor ion, is required. In this thesis, electrospray ionization multistage tandem mass spectrometry (ESI-MS n) was employed to examine the non-covalent complexes between poly(styrene sulfonate) (PSS) and poly-L-lysine (PLL). During single-stage ion activation, the PLL peptide chain mainly underwent backbone cleavages without disruption of the noncovalent interaction which could only be broken via sequential application of electron transfer dissociation (ETD) and collisionally activated dissociation (CAD), indicating strong binding interactions between the two polyelectrolyte chains. Such binding properties make PSS a potential "non-covalent (supramolecular) label" for determining the surface accessibility of basic residues on a peptide or protein. To probe this premise, noncovalent complexes of substance P and PSS were characterized by ESI-MS n using different ion activation methods. Both MS 2 and MS 3 experiments on the substance P + PSS complex resulted in the formation of bn (on CAD) or cn (on ETD) fragments attached non-covalently to the intact PSS chain. All peptide fragments containing the intact PSS chain included Arg1, Lys3, and Gln5, pointing out that these residues, which are located near the Nterminus, are most likely involved in the noncovalent interaction with PSS. In contrast, iv Gln6 was excluded from this fragment series, attesting a much weaker interaction with PSS due to lesser accessibility. The strong tendency of PSS to bind peptides noncovalently at sites that can be elucidated by MS n demonstrates a proof-of-concept for the capacity of this approach to unveil higher order structure in proteins. v ACKNOWLEDGEMENTS I would like to first express my deepest gratitude to my advisor, Dr. Chrys Wesdemiotis for his excellent guidance, valuable suggestions and kindness during the process of my M.S. studies at the University of Akron. His knowledge in chemistry and mass spectrometry directed me towards the right path to achieve my goals during these two years. I would also like to thank him for his help and suggestions regarding my future studies. I would like to thank my committee member Dr. Toshikazu Miyoshi for taking his time to read my thesis and giving me great advice and feedback. He is such a nice person that I do not hesitate to ask for help whenever I have difficulties. I am also grateful for the Department of Polymer Science at The University of Akron. Thanks for holding such an excellent program to support me to do research in such a great environment. The faculty and staff here are all so kind to make me feel at home.
Affinity surface-assisted laser desorption/ionization mass spectrometry for peptide enrichment
The Analyst, 2012
In this paper, we report on the functionalization of silicon nanostructured (NanoSi) surface with an organic layer of nitrilotriacetic acid (NTA) and its subsequent use as an affinity surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) interface for histidine-tagged peptide enrichment and mass spectrometry analysis. The NTA terminal groups are immobilized onto the NanoSi surface via very stable Si-C covalent bonds. The NTA-modified NanoSi (NTA-NanoSi) interface was characterized by contact angle measurements, Fourier transform infrared (FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS). The NTA-NanoSi interface has shown a good selectivity toward His-tagged peptide and permits its enrichment from an artificial mixture of both tagged and untagged peptides and its subsequent mass spectrometry detection with good signal/noise ratio.
Positively charged peptides can interact with each other, as revealed by solid phase binding assays
Analytical Biochemistry, 2006
Solid phase assay systems such as enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), and overlay gels are used to study processes of protein-protein interactions. The common principle of all these methods is that they monitor the binding between soluble and surface-immobilized molecules. Following the use of bovine serum albumin (BSA)-peptide conjugates or isolated synthetic peptides and the above-mentioned solid phase assay systems, the results of the current work demonstrate that positively charged peptides can interact with each other. Both the ELISA and SPR methods demonstrated that the binding process reached saturation with K d values ranging between 1 and 14 nM. No interaction was observed between BSA conjugates bearing positively charged peptides and conjugates bearing negatively charged peptides or with pure BSA molecules, strengthening the view that interaction occurs only between positively charged peptides. However, interactions between peptides in solution were not observed by nuclear magnetic resonance (NMR) or by native gel electrophoresis. It appears that for positively charged molecules to interact, one of the binding partners must be immobilized to a surface, a process that may lead to the exposure of otherwise masked groups or atoms. We discuss the relevance of our Wndings for the use of solid phase assay systems to study interactions between biomolecules.
Laser desorption ionization mass spectrometry of peptides on a hybrid CHCA organic–inorganic matrix
The Analyst, 2014
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Analytical Chemistry, 2004
Chemical derivatization of peptides allows efficient F 2 laser single photon ionization (SPI) of Fmoc-derivatized peptides covalently bound to surfaces. Laser desorption photoionization mass spectrometry using 337-nm pulses for desorption and 157.6-nm pulses for threshold SPI forms large ions identified as common peptide fragments bound to either Fmoc or the surface linker. Electronic structure calculations indicate the Fmoc label is behaving as an ionization tag for the entire peptide, lowering the ionization potential of the complex below the 7.87-eV photon energy. This method should allow detection of many molecular species covalently or electrostatically bound to surfaces.