Structure elucidation of tertiary or quaternary protein structures by chemical cross-linking and mass spectrometry (MS) has recently gained importance. To locate the cross-linker modification, dedicated software is applied to analyze the mass or tandem mass spectra (MS/MS). Such software requires information on target amino acids to limit the data analysis time. The most commonly used homobifunctional N-hydroxy succinimide (NHS) esters are often described as reactive exclusively towards primary amines, although side reactions with tyrosine and serine have been reported. Our goal was to systematically study the reactivity of NHS esters and derive some general rules for their attack of nucleophilic amino acid side chains in peptides. We therefore studied the cross-linking reactions of synthesized and commercial model peptides with disuccinimidyl suberate (DSS). The first reaction site in all cases was expectedly the alpha-NH(2)-group of the N-terminus or the epsilon-NH(2)-group of lysine. As soon as additional cross-linkers were attached or loops were formed, other amino acids were also involved in the reaction. In addition to the primary amino groups, serine, threonine and tyrosine showed significant reactivity due to the effect of neighboring amino acids by intermediate or permanent Type-1 cross-link formation. The reactivity is highly dependent on the pH and on adjacent amino acids.
Functional high-density micro-arrays for mass spectrometry enable rapid picolitre-volume aliquoting and ultrasensitive analysis of microscale samples, for example, single cells.
The heme protein, cytochrome c (Cc) has been studied using tip-enhanced Raman spectroscopy (TERS). By virtue of its sensitivity and superior spatial resolution, TERS detected both the heme and amino acid vibrational bands of Cc using resonance excitation at 532 nm. This is in contrast to conventional surface-enhanced Raman spectroscopy (SERS) where ensemble information is obtained, leading to the strongest Raman bands obscuring the weaker ones; i.e., only the resonantly enhanced heme bands are observed. Matrix-assisted laser desorption/ ionization mass spectrometry supported the interpretation of the Raman data by showing that Cc remains intact on Ag surfaces. This work demonstrates the sensitivity of TERS for analyzing proteins (complete with band assignments) and that a collection of TER spectra gives a more complete description of large biomolecules. The latter is an important advantage usually not found in SERS measurements.
IntroductionIn the present study, we sought to quantify and contrast the secretome and biomechanical properties of the non-chondrodystrophic (NCD) and chondrodystrophic (CD) canine intervertebral disc (IVD) nucleus pulposus (NP).MethodsWe used iTRAQ proteomic methods to quantify the secretome of both CD and NCD NP. Differential levels of proteins detected were further verified using immunohistochemistry, Western blotting, and proteoglycan extraction in order to evaluate the integrity of the small leucine-rich proteoglycans (SLRPs) decorin and biglycan. Additionally, we used robotic biomechanical testing to evaluate the biomechanical properties of spinal motion segments from both CD and NCD canines.ResultsWe detected differential levels of decorin, biglycan, and fibronectin, as well as of other important extracellular matrix (ECM)-related proteins, such as fibromodulin and HAPLN1 in the IVD NP obtained from CD canines compared with NCD canines. The core proteins of the vital SLRPs decorin and biglycan were fragmented in CD NP but were intact in the NP of the NCD animals. CD and NCD vertebral motion segments demonstrated significant differences, with the CD segments having less stiffness and a more varied range of motion.ConclusionsThe CD NP recapitulates key elements of human degenerative disc disease. Our data suggest that at least some of the compromised biomechanical properties of the degenerative disc arise from fibrocartilaginous metaplasia of the NP secondary to fragmentation of SLRP core proteins and associated degenerative changes affecting the ECM. This study demonstrates that the degenerative changes that naturally occur within the CD NP make this animal a valuable animal model with which to study IVD degeneration and potential biological therapeutics.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0733-z) contains supplementary material, which is available to authorized users.
Chemical cross-linking in combination with mass spectrometry has emerged as a powerful tool to study noncovalent protein complexes. Nevertheless, there are still many questions to answer. Does the amount of detected cross-linked complex correlate with the amount of protein complex in solution? In which concentration and affinity range is specific cross-linking possible? To answer these questions, we performed systematic cross-linking studies with two complexes, using the N-hydroxysuccinimidyl ester disuccinimidyl suberate (DSS): (1) NCoA-1 and mutants of the interacting peptide STAT6Y, covering a K D range of 30 nM to Ͼ25 M, and (2) ␣-thrombin and basic pancreatic trypsin inhibitor (BPTI), a system that shows a bufferdependent K D value between 100 and 320 M. Samples were analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). For NCoA-1•STAT6Y, a good correlation between the amount of cross-linked species and the calculated fraction of complex present in solution was observed. Thus, chemical cross-linking in combination with MALDI-MS can be used to rank binding affinities. For the mid-affinity range up to about K D Ϸ 25 M, experiments with a nonbinding peptide and studies of the concentration dependence showed that only specific complexes undergo cross-linking with DSS. To study in which affinity range specific cross-linking can be applied, the weak ␣-thrombin•BPTI complex was investigated. We found that the detected complex is a nonspecifically cross-linked species. Consequently, based on the experimental approach used in this study, chemical cross-linking is not suitable for studying low-affinity complexes with K D ӷ 25 M. (J Am Soc Mass Spectrom 2010Spectrom , 21, 1775Spectrom -1783
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