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1.
Tingting Fu Janina Oetjen Manuel Chapelle Alexandre Verdu Matthias Szesny Arnaud Chaumot Davide Degli‐Esposti Olivier Geffard Yohann Clment Arnaud Salvador Sophie Ayciriex 《Journal of mass spectrometry : JMS》2020,55(9)
The highly diverse chemical structures of lipids make their analysis directly from biological tissue sections extremely challenging. Here, we report the in situ mapping and identification of lipids in a freshwater crustacean Gammarus fossarum using matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) in combination with an additional separation dimension using ion mobility spectrometry (IMS). The high‐resolution trapped ion mobility spectrometry (TIMS) allowed efficient separation of isobaric/isomeric lipids showing distinct spatial distributions. The structures of the lipids were further characterized by MS/MS analysis. It is demonstrated that MALDI MSI with mobility separation is a powerful tool for distinguishing and localizing isobaric/isomeric lipids. 相似文献
2.
Ruben Ceuppens Debora Dumont Leen Van Brussel Babs Van de Plas Ruth Daniels Jean-Paul Noben Peter Verhaert Estel Van der Gucht Johan Robben Stefan Clerens Lutgarde Arckens 《International journal of mass spectrometry》2007,260(2-3):185
One of the newly developed imaging mass spectrometry (IMS) technologies utilizes matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to map proteins in thin tissue sections. In this study, we evaluated the power of MALDI IMS as we developed it in our (Bruker) MALDI TOF (Reflex IV) and TOF-TOF (Ultraflex II) systems to study myelin patterns in the mouse central nervous system under normal and pathological conditions. MALDI IMS was applied to assess myelin basic protein (MBP) isoform-specific profiles in different regions throughout the mouse brain. The distribution of ions of m/z 14,144 and 18,447 displayed a striking resemblance with white matter histology and were identified as MBP isoform 8 and 5, respectively. In addition, we demonstrated a significant reduction of the MBP-8 peak intensity upon MALDI IMS analysis of focal ethidium bromide-induced demyelinated brain areas. Our MS images were validated by immunohistochemistry using MBP antibodies. This study underscores the potential of MALDI IMS to study the contribution of MBP to demyelinating diseases. 相似文献
3.
Trypsin and MALDI matrix pre‐coated targets simplify sample preparation for mapping proteomic distributions within biological tissues by imaging mass spectrometry
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Faizan Zubair Paul E. Laibinis William G. Swisher Junhai Yang Jeffrey M. Spraggins Jeremy L. Norris Richard M. Caprioli 《Journal of mass spectrometry : JMS》2016,51(12):1168-1179
Prefabricated surfaces containing α‐cyano‐4‐hydroxycinnamic acid and trypsin have been developed to facilitate enzymatic digestion of endogenous tissue proteins prior to matrix‐assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS). Tissue sections are placed onto slides that were previously coated with α‐cyano‐4‐hydroxycinnamic acid and trypsin. After incubation to promote enzymatic digestion, the tissue is analyzed by MALDI IMS to determine the spatial distribution of the tryptic fragments. The peptides detected in the MALDI IMS dataset were identified by Liquid chromatography‐tandem mass spectrometry/mass spectrometry. Protein identification was further confirmed by correlating the localization of unique tryptic fragments originating from common parent proteins. Using this procedure, proteins with molecular weights as large as 300 kDa were identified and their distributions were imaged in sections of rat brain. In particular, large proteins such as myristoylated alanine‐rich C‐kinase substrate (29.8 kDa) and spectrin alpha chain, non‐erythrocytic 1 (284 kDa) were detected that are not observed without trypsin. The pre‐coated targets simplify workflow and increase sample throughput by decreasing the sample preparation time. Further, the approach allows imaging at higher spatial resolution compared with robotic spotters that apply one drop at a time. Copyright © 2016 John Wiley & Sons, Ltd. 相似文献
4.
Maolei Zhu Brad Bendiak Brian Clowers Herbert H. Hill Jr 《Analytical and bioanalytical chemistry》2009,394(7):1853-1867
The rapid separation of isomeric precursor ions of oligosaccharides prior to their analysis by mass spectrometry to the nth power (MS
n
) was demonstrated using an ambient pressure ion mobility spectrometer (IMS) interfaced with a quadrupole ion trap. Separations
were not limited to specific types of isomers; representative isomers differing solely in the stereochemistry of sugars, in
their anomeric configurations, and in their overall branching patterns and linkage positions could be resolved in the millisecond
time frame. Physical separation of precursor ions permitted independent mass spectra of individual oligosaccharide isomers
to be acquired to at least MS3, the number of stages of dissociation limited only practically by the abundance of specific product ions. IMS–MS
n
analysis was particularly valuable in the evaluation of isomeric oligosaccharides that yielded identical sets of product
ions in tandem mass spectrometry experiments, revealing pairs of isomers that would otherwise not be known to be present in
a mixture if evaluated solely by MS dissociation methods alone. A practical example of IMS–MSn analysis of a set of isomers included within a single high-performance liquid chromatography fraction of oligosaccharides
released from bovine submaxillary mucin is described. 相似文献
5.
Zimmerman TA Rubakhin SS Sweedler JV 《Journal of the American Society for Mass Spectrometry》2011,22(5):828-836
Mass spectrometry imaging (MSI) provides the ability to detect and identify a broad range of analytes and their spatial distributions
from a variety of sample types, including tissue sections. Here we describe an approach for probing neuropeptides from sparse
cell cultures using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MSI—at single cell spatial resolution—in
both MS and tandem MS modes. Cultures of Aplysia californica neurons are grown on an array of glass beads embedded in a stretchable layer of Parafilm M. As the membrane is stretched,
the beads/neurons are separated physically and the separated beads/neurons analyzed via MALDI TOF MS. Compared with direct
MS imaging of samples, the stretching procedure enhances analyte extraction and incorporation into the MALDI matrix, with
negligible analyte spread between separated beads. MALDI tandem MSI using the stretched imaging approach yields localization
maps of both parent and fragment ions from Aplysia pedal peptide, thereby confirming peptide identification. This methodology represents a flexible platform for MSI investigation
of a variety of cell cultures, including functioning neuronal networks. 相似文献
6.
Melanie Jünger Bertram Bödeker Jörg Ingo Baumbach 《Analytical and bioanalytical chemistry》2010,396(1):471-482
Over the past years, ion mobility spectrometry (IMS) as a well established method within the fields of military and security
has gained more and more interest for biological and medical applications. This highly sensitive and rapid separation technique
was crucially enhanced by a multi-capillary column (MCC), pre-separation for complex samples. In order to unambiguously identify
compounds in a complex sample, like breath, by IMS, a reference database is mandatory. To obtain a first set of reference
data, 16 selected volatile organic substances were examined by MCC-IMS and comparatively analyzed by the standard technique
for breath research, thermal desorption–gas chromatography–mass spectrometry. Experimentally determined MCC and GC retention
times of these 16 compounds were aligned and their relation was expressed in a mathematical function. Using this function,
a prognosis of the GC retention time can be given very precisely according to a recorded MCC retention time and vice versa.
Thus, unknown MCC-IMS peaks from biological samples can be assigned—after alignment via the estimated GC retention time—to
analytes identified by GC/MS from equivalent accomplished data. One example of applying the peak assignment strategy to a
real breath sample is shown in detail. 相似文献
7.
Groseclose MR Andersson M Hardesty WM Caprioli RM 《Journal of mass spectrometry : JMS》2007,42(2):254-262
A novel method for on-tissue identification of proteins in spatially discrete regions is described using tryptic digestion followed by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) with MS/MS analysis. IMS is first used to reveal the protein and peptide spatial distribution in a tissue section and then a serial section is robotically spotted with small volumes of trypsin solution to carry out in situ protease digestion. After hydrolysis, 2,5-Dihydroxybenzoic acid (DHB) matrix solution is applied to the digested spots, with subsequent analysis by IMS to reveal the spatial distribution of the various tryptic fragments. Sequence determination of the tryptic fragments is performed using on-tissue MALDI MS/MS analysis directly from the individual digest spots. This protocol enables protein identification directly from tissue while preserving the spatial integrity of the tissue sample. The procedure is demonstrated with the identification of several proteins in the coronal sections of a rat brain. 相似文献
8.
Daniel Pröfrock 《Analytical and bioanalytical chemistry》2010,398(6):2383-2401
The application of miniaturised separation techniques such as capillary LC, nano LC or capillary electrophoresis offers a
number of advantages in terms of analytical performance, solvent consumption and the ability to analyse very small sample
amounts. These features make them attractive for various bioanalytical tasks, in particular those related to the analysis
of proteins and peptides. The skillful combination of such techniques with inductively coupled plasma mass spectrometry (ICP-MS)
has recently permitted the design of combined analytical approaches utilising either elemental or molecule-specific detection
techniques such as electrospray ionisation (ESI) or matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry
in a highly complementary manner for, as an example, proteomics-orientated research (heteroatom-tagged proteomics). Such hybrid
approaches are, in particular, providing promising new options for the fast screening of complex samples for specific metal-containing
or—more generally speaking—heteroatom-containing biomolecules, as well as the accurate absolute quantification of biomolecules,
which is still an unsolved problem in bioanalysis. Here, progress in as well as the potential and the special requirements
of hyphenating miniaturised separation techniques with ICP-MS are reviewed and critically discussed. In addition, selected
applications are highlighted to indicate current and possible future trends within this emerging area of research. 相似文献
9.
Hayasaka T Goto-Inoue N Ushijima M Yao I Yuba-Kubo A Wakui M Kajihara S Matsuura M Setou M 《Analytical and bioanalytical chemistry》2011,401(1):183-193
Imaging mass spectrometry (IMS) is a powerful tool for detecting and visualizing biomolecules in tissue sections. The technology
has been applied to several fields, and many researchers have started to apply it to pathological samples. However, it is
very difficult for inexperienced users to extract meaningful signals from enormous IMS datasets, and the procedure is time-consuming.
We have developed software, called IMS Convolution with regions of interest (ROI), to automatically extract meaningful signals
from IMS datasets. The processing is based on the detection of common peaks within the ordered area in the IMS dataset. In
this study, the IMS dataset from a mouse eyeball section was acquired by a mass microscope that we recently developed, and
the peaks extracted by manual and automatic procedures were compared. The manual procedure extracted 16 peaks with higher
intensity in mass spectra averaged in whole measurement points. On the other hand, the automatic procedure using IMS Convolution
easily and equally extracted peaks without any effort. Moreover, the use of ROIs with IMS Convolution enabled us to extract
the peak on each ROI area, and all of the 16 ion images on mouse eyeball tissue were from phosphatidylcholine species. Therefore,
we believe that IMS Convolution with ROIs could automatically extract the meaningful peaks from large-volume IMS datasets
for inexperienced users as well as for researchers who have performed the analysis. 相似文献
10.
Wu S Zhang L Yang K Liang Z Zhang L Zhang Y 《Analytical and bioanalytical chemistry》2012,402(2):703-710
Initially, a poly (glycidyl methacrylate-co-acrylamide-co-methylenebisacrylamide) monolith was prepared in the 100 μm i.d.
capillary, and then was grafted with polyethylenimine (Mw, ∼25,000) for adsorbing Cu2+, followed by chelating trypsin. As a result, efficient digestion for BSA (100 ng/μL) was completed within 50 s via such immobilized
enzyme reactor (IMER); yielding 47% sequence coverage by matrix-assisted laser desorption/ionization-time of flight mass spectrometry
(MALDI-TOF MS) analysis. Compared with the conventional method for preparing the metal-ion chelated IMER, the regeneration
of such IMER can be achieved facilely by the respective 30 min desorption and re-adsorption of trypsin, and 51% sequence coverage
was obtained for 50 s BSA digestion after regeneration. BSA down to femtomole was also efficiently digested by the prepared
regenerable IMER. Meanwhile, after the consecutive digestion of myoglobin and BSA, there was not any mutual interference for
both during MALDI-TOF MS identification, indicating the low nonspecific adsorption of such regenerable IMER. To test the applicability
of regenerable IMER for complex sample profiling, proteins (150 ng) extracted from Escherichia coli were digested within 80 s by the regenerable IMER and further analyzed by nanoreversed phase liquid chromatography–electrospray
ionization–mass spectrometry successfully, showing its practicability for the high throughput analysis of complex samples. 相似文献
11.
Chong SL Nissilä T Ketola RA Koutaniemi S Derba-Maceluch M Mellerowicz EJ Tenkanen M Tuomainen P 《Analytical and bioanalytical chemistry》2011,401(9):2995-3009
The atmospheric pressure matrix-assisted laser desorption/ionization with ion trap mass spectrometry (AP-MALDI-ITMS) was investigated
for its ability to analyse plant-derived oligosaccharides. The AP-MALDI-ITMS was able to detect xylooligosaccharides (XOS)
with chain length of up to ten xylopyranosyl residues. Though the conventional MALDI–time-of-flight/mass spectrometry (TOF/MS)
showed better sensitivity at higher mass range (>m/z 2,000), the AP-MALDI-ITMS seems to be more suitable for detection of acetylated XOS, and the measurement also corresponded
better than the MALDI-TOF/MS analysis to the actual compositions of the pentose- and hexose-derived oligosaccharides in a
complex sample. The structures of two isomeric aldotetrauronic acids and a mixture of acidic XOS were elucidated by AP-MALDI-ITMS
using multi-stages mass fragmentation up to MS3. Thus, the AP-MALDI-ITMS demonstrated an advantage in determining both mass and structures of plant-derived oligosaccharides.
In addition, the method of combining the direct endo-1,4-β-d-xylanase hydrolysis of plant material, and then followed by AP-MALDI-ITMS detection, was shown to recognize the substitution
variations of glucuronoxylans in hardwood species and in Arabidopsis thaliana. To our knowledge, this is the first report to demonstrate the acetylation of glucuronoxylan in A. thaliana. The method, which requires only a small amount of plant material, such as 1 to 5 mg for the A. thaliana stem material, can be applied as a high throughput fingerprinting tool for the fast comparison of glucuronoxylan structures
among plant species or transformants that result from in vivo cell wall modification. 相似文献
12.
Matrix‐assisted laser/desorption ionization imaging mass spectrometry (MALDI IMS) is an analytical technique for understanding the spatial distribution of biomolecules across a sample surface. Originally employed for mammalian tissues, this technology has been adapted to study specimens as diverse as microbes and cell cultures, food such as strawberries, and invertebrates including the vinegar fly Drosophila melanogaster. As an ideal model organism, Drosophila has brought greater understanding about conserved biological processes, organism development, and diseased states and even informed management practices of agriculturally and environmentally important species. Drosophila displays anatomically separated renal (Malpighian) tubules that are the physiological equivalent to the vertebrate nephron. Insect Malpighian tubules are also responsible for pesticide detoxification. In this article, we first describe an effective workflow and sample preparation method to study the phospholipid distribution of the Malpighian tubules that initially involves the manual microdissection of the tubules in saline buffer followed by a series of washes to remove excess salt and enhances the phospholipid signals prior to matrix deposition and IMS at 25‐μm spatial resolution. We also established a complementary methodology for lipid IMS analysis of whole‐body fly sections using a dual‐polarity data acquisition approach at the same spatial resolution after matrix deposition by sublimation. Both procedures yield rich signal profiles from the major phospholipid classes. The reproducibility and high‐quality results offered by these methodologies enable cohort studies of Drosophila through MALDI IMS. 相似文献
13.
Mirela Sarbu Dragana Fabris
eljka Vukeli David E. Clemmer Alina D. Zamfir 《Molecules (Basel, Switzerland)》2022,27(3)
Gangliosides (GGs) represent an important class of biomolecules associated with the central nervous system (CNS). In view of their special role at a CNS level, GGs are valuable diagnostic markers and prospective therapeutic agents. By ion mobility separation mass spectrometry (IMS MS), recently implemented by us in the investigation of human CNS gangliosidome, we previously discovered a similarity between GG profiles in CSF and the brain. Based on these findings, we developed IMS tandem MS (MS/MS) to characterize rare human CSF glycoforms, with a potential biomarker role. To investigate the oligosaccharide and ceramide structures, the ions detected following IMS MS separation were submitted to structural analysis by collision-induced dissociation (CID) MS/MS in the transfer cell. The IMS evidence on only one mobility feature, together with the diagnostic fragment ions, allowed the unequivocal identification of isomers in the CSF. Hence, by IMS MS/MS, GalNAc-GD1c(d18:1/18:1) and GalNAc-GD1c(d18:1/18:0) having both Neu5Ac residues and GalNAc attached to the external galactose were for the first time discovered and structurally characterized. The present results demonstrate the high potential of IMS MS/MS for biomarker discovery and characterization in body fluids, and the perspectives of method implementation in clinical analyses targeting the early diagnosis of CNS diseases through molecular fingerprints. 相似文献
14.
Jackson SN Ugarov M Post JD Egan T Langlais D Schultz JA Woods AS 《Journal of the American Society for Mass Spectrometry》2008,19(11):1655-1662
Combining matrix-assisted laser desorption/ionization (MALDI) mass spectrometry with ion mobility (IM) results in the fast
sorting of biomolecules in complex mixtures along trend lines. In this two-dimensional (2D) analysis of biological families,
lipids, peptides, and nucleotides are separated from each other by differences in their ion mobility drift times in a timescale
of hundreds of microseconds. Molecular ions of similar chemical type fall along trend lines when plotted in 2D plots of ion
mobility drift time as a function of m/z. In this study, MALDI-IM MS is used to analyze species from all of the major phospholipid classes. Complex samples, including
tissue extracts and sections, were probed to demonstrate the effects that radyl chain length, degree of unsaturation, and
class/head group have upon an ion’s cross section in the gas phase. We illustrate how these changes can be used to identify
individual lipid species in complex mixtures, as well as the effects of cationization on ion cross section and ionization
efficiency. 相似文献
15.
Kubo A Ohmura M Wakui M Harada T Kajihara S Ogawa K Suemizu H Nakamura M Setou M Suematsu M 《Analytical and bioanalytical chemistry》2011,400(7):1895-1904
Analyses of energy metabolism in human cancer have been difficult because of rapid turnover of the metabolites and difficulties
in reducing time for collecting clinical samples under surgical procedures. Utilization of xenograft transplantation of human-derived
colon cancer HCT116 cells in spleens of superimmunodeficient NOD/SCID/IL-2Rγnull (NOG) mice led us to establish an experimental model of hepatic micrometastasis of the solid tumor, whereby analyses of the
tissue sections collected by snap-frozen procedures through newly developed microscopic imaging mass spectrometry (MIMS) revealed
distinct spatial distribution of a variety of metabolites. To perform intergroup comparison of the signal intensities of metabolites
among different tissue sections collected from mice in fed states, we combined matrix-assisted laser desorption/ionization
time-of-flight imaging mass spectrometry (MALDI–TOF-IMS) and capillary electrophoresis–mass spectrometry (CE–MS), to determine
the apparent contents of individual metabolites in serial tissue sections. The results indicated significant elevation of
ATP and energy charge in both metastases and the parenchyma of the tumor-bearing livers. To note were significant increases
in UDP-N-acetyl hexosamines, and reduced and oxidized forms of glutathione in the metastatic foci versus the liver parenchyma. These
findings thus provided a potentially important method for characterizing the properties of metabolic systems of human-derived
cancer and the host tissues in vivo. 相似文献
16.
Laurence Charles Christophe Chendo Salom Poyer 《Rapid communications in mass spectrometry : RCM》2020,34(Z2)
This review covers applications of ion mobility spectrometry (IMS) hyphenated to mass spectrometry (MS) in the field of synthetic polymers. MS has become an essential technique in polymer science, but increasingly complex samples produced to provide desirable macroscopic properties of high‐performance materials often require separation of species prior to their mass analysis. Similar to liquid chromatography, the IMS dimension introduces shape selectivity but enables separation at a much faster rate (milliseconds vs minutes). As a post‐ionization technique, IMS can be hyphenated to MS to perform a double separation dimension of gas‐phase ions, first as a function on their mobility (determined by their charge state and collision cross section, CCS), then as a function of their m/z ratio. Implemented with a variety of ionization techniques, such coupling permits the spectral complexity to be reduced, to enhance the dynamic range of detection, or to achieve separation of isobaric ions prior to their activation in MS/MS experiments. Coupling IMS to MS also provides valuable information regarding the 3D structure of polymer ions in the gas phase and regarding how to address the question of how charges are distributed within the structure. Moreover, the ability of IMS to separate multiply charged species generated by electrospray ionization yields typical IMS‐MS 2D maps that permit the conformational dynamics of synthetic polymer chains to be described as a function of their length. 相似文献
17.
Hayasaka T Goto-Inoue N Sugiura Y Zaima N Nakanishi H Ohishi K Nakanishi S Naito T Taguchi R Setou M 《Rapid communications in mass spectrometry : RCM》2008,22(21):3415-3426
We recently developed a matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight (MALDI-QIT-TOF)-based imaging mass spectrometry (IMS) system. This system enables us to perform structural analyses using tandem mass spectrometry (MS/MS), as well as to visualize phospholipids and peptides in frozen sections. In the retina, phototransduction is regulated by the light-sensitive interaction between visual pigment-coupled receptor proteins, such as rhodopsin, and G proteins, such as transducin. There are some reports that the conformation of rhodopsin is influenced by the composition of phospholipids in the lipid bilayer membrane. However, these results were based on in vitro experiments and have not been analyzed in vivo. In this study, we visualized and identified phospholipids in mouse retinal sections with the MALDI-QIT-TOF-based IMS system. From a spectrum obtained by raster-scanned analysis of the sections, ions with high signal intensities were selected and analyzed by MS/MS. As a result, sixteen ions were identified as being from four diacyl-phosphatidylcholine (PC) species, i.e., PC (16:0/16:0), PC (16:0/18:1), PC (16:0/22:6), and PC (18:0/22:6), with different ion forms. The ion images revealed different distributions on the retinal sections: PC (16:0/18:1) was distributed in the inner nuclear layer and outer plexiform layer, PC (16:0/16:0) in the outer nuclear layer and inner segment, and both PC (16:0/22:6) and PC (18:0/22:6) in the outer segment and pigment epithelium. In conclusion, our in vivo IMS analyses demonstrated a three-zone distribution of PC species on the retinal sections. This approach may be useful for analyzing lipid changes and their contribution to phototransduction in the retina. 相似文献
18.
Recently, we developed a method for modified ribonucleic acid (RNA) analysis based on the comparative analysis of RNA digests (CARD). Within this CARD approach, sequence or modification differences between two samples are identified through differential isotopic labeling of two samples. Components present in both samples will each be labeled, yielding doublets in the CARD mass spectrum. Components unique to only one sample should be detected as singlets. A limitation of the prior singlet identification strategy occurs when the two samples contain components of unique sequence but identical base composition. At the first stage of mass spectrometry, these sequence isomers cannot be differentiated and would appear as doublets rather than singlets. However, underlying sequence differences should be detectable by collision‐induced dissociation tandem mass spectrometry (CID MS/MS), as y‐type product ions will retain the original enzymatically incorporated isotope label. Here, we determine appropriate instrumental conditions that enable CID MS/MS of isotopically labeled ribonuclease T1 (RNase T1) digestion products such that the original isotope label is maintained in the product ion mass spectrum. Next, we demonstrate how y‐type product ions can be used to differentiate singlets and doublets from isomer sequences. We were then able to extend the utility of this approach by using CID MS/MS for the confirmation of an expected RNase T1 digestion product within the CARD analysis of an Escherichia coli mutant strain even in the presence of interfering and overlapping digestion products from other transfer RNAs. Copyright © 2014 John Wiley & Sons, Ltd. 相似文献
19.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was optimized to discriminate
between wild-type and ampicillin-resistant Escherichia coli. Only ampicillin-resistant E. coli displayed an m/z ≈ 29,000 peak, which was confirmed as β-lactamase by in-gel digestion followed by peptide mass fingerprinting. Rapid MALDI-TOF
MS detection of antibiotic-resistance could fulfill an important clinical need, providing critical phenotypic information
beyond genus–species identification. 相似文献
20.
Protein—cisplatin interactions lie at the heart of both the effectiveness of cisplatin as a therapeutic agent and side effects
associated with cisplatin treatment. Because a greater understanding of the protein—cisplatin interactions at the molecular
level can inform the design of cisplatin-like agents for future use, mass spectrometric determination of the binding site
of cisplatin on a model protein, cytochrome c, was undertaken in this paper. The monoadduct cytochrome c—Pt(NH3)2(H2O) is found to be the primary adduct produced by the cytochrome c—cisplatin interactions under native conditions. To locate the primary binding site of cisplatin, both free cytochrome c and the cytochrome c adducts underwent trypsin digestion, followed by Fourier transform mass spectrometry (FT-MS) to identify unique fragments
in the adduct digest. Four such fragments were found in the adduct digest. Tandem mass spectrometry (MS/MS and MS3 indicates that two fragments are Pt(NH3)2(H2O) bound peptides (Gly56-Glu104 and Asn54-Glu104) with one water associated at the peptide bond Lys79∼Met80, and the other
two fragments are heme containing peptides (acety1-Gly1-Lys53 and acety1-Gly1-Lys55). The product-ion spectra of the four
fragments reveal that Met65 is the primary binding site of cisplatin on cytochrome c. 相似文献