Recently, I try to obtain disaccharide peptides and lactoyl peptides from Mycobacterium Smegmatis, but I cannot succeed. My method is:
Disaccharide peptides: The mycobacterial strains were disrupted by Low Temperature Ultra-high Uressure Continuous Flow Cell Disrupter. The cell suspension was treated with DNase and RNase. Then the pellet was resuspended in PBS with 4% SDS and incubated at boiling water for 30 min. The pellet was further treated with pronase, trypsin, lysozyme and mutanolysin. The soluble disaccharide peptides were recovered by ultracentrifugation.
Lactoyl peptides: To the solution of 200 μl of disaccharide peptides was added 30% ammonium hydroxide (68 μl), and the reaction mixture was incubated at 37°C for 5 h. The reaction was neutralized with 61 μl of acetic acid, lyophilized, and dissolved in water containing 0.05% TFA.
Purification: The lactoyl peptides were separated by a HPLC system. Lactoyl peptides were applied on the C18 column and the muropeptides were eluted in 30% acetonitrile (containing 0.05% TFA) at a flow rate of 0.5 ml/min. The materials eluted in the individual peaks were collected separately, lyophilized, and dissolved in water for MALDI-TOF analysis.
However, my data of MALDI-TOF analysis did not have known mass according to literature (e.g. [M+H]+ ion at m/z 974.5, 532.3, 1021.5 and so on).
So, I hope who knows a specific protocol to prepare for disaccharide peptides and lactoyl peptides of Mycobacteria could tell me my mistakes or notes. Thanks!
Mr. Xiaoyu,
I shall discuss only the part of your research tasks associated with MALDI-MS analysis of carbohydrates, due to:
(i) We have a significant experience with mass spectrometric analysis of the chemistry of carbohydrates; and
(ii) we have developed more recently our own-authored (to me and my co-author) mass spectrometric method based on stochastic dynamics with corresponding innovative model equations, which provide exact (or absolute) direct quantitative information about analytes in solution; as well as 3D molecular conformational and electronic stuctural information about the analytes; which further is applicable to MALDI-MS method.
Let us start, first, with the fact that the chemistry of carbohydrates in solution is very complex yielding to different oligomeric and macromolecular products depending on the experimental conditions.
Furthermore, under MALDI-MS ionization conditions, there are a set of additional processes of ionization associated with a formation of metal-organic species in presence of alkali metal ions or NH4+-ion.
Therefore, the fact that your MALDI-MS spectrum does not fit to available literature data could be a result from different experimental conditions.
Please, consider, few of our contributions devoted to mass spectrometry of the chemistry of carbohydrates [1-3].
[1] International Journal of Biological Macromolecules, 87 (2016) 263-272
Molecular and environmental factors governing non–covalent bonding interactions and conformations of phosphorous functionalized γ-cyclodextrin hydrate systems; Bojidarka Ivanova, Michael Spiteller
[2] International Journal of Biological Macromolecules, 64 (2014) 383-391
Macromolecular ensembles of cyclodextrin crystallohydrates and clathrates – experimental and theoretical gas – and condense phase study; Bojidarka Ivanova, Michael Spiteller
[3] Bioorganic Chemistry, 93 (2019) 103308
A mass spectrometric stochastic dynamic diffusion approach to selective quantitative and 3D structural analyses of native cyclodextrins by electrospray ionization and atmospheric pressure chemical ionization methods; Bojidarka Ivanova, Michael Spiteller
Thus, the chemistry of carbohydrates, as mentioned before, is very complex and the mass spectrometric analysis of these analytes does not represent a trivial research task, in general.
Second, reference [4] deals with our aforementioned innovative mass spectrometric theory and model equations, which provide not only exact analytical quantitative and 3D structural information about the analytes, but also it appears universally applicable to different ionization methods. For instance, these are ESI, APCI, and CID methods, as well as MALDI-MS one.
Despite, reference [4] illustrates the capability of our innovative theory to APCI-MS data, it is applicable to MALDI-MS ones, as well. In the latter context, please consider reference [5].
[4] Steroids, 164 (2020) 108750
Stochastic dynamic mass spectrometric quantification of steroids in mixture — Part II; Bojidarka Ivanova, Michael Spiteller
[5] Bojidarka Ivanova and Michael Spiteller, Quantification by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry Using An Approach Based On Stochastic Dynamics. Experimental And Theoretical Correspondences; GRIN Verlag, Muenchen, (2018) ISBN: 9783668703162; pp. 1-83; [https://www.grin.com/document/425061].
Finally, looking at the complexity of your microbiological object, I should explicitly emphasize, that valuable, reliable, precise and accurate mass spectrometric assignment of chemical reactions of carbohydrates, even, talking about simple mono- or disaccharides; as well as, their MS both quantitative exact determination, or obtaining of exact 3D molecular conformation or electronic structures compulsory require the task of the so-called ''back-synthesis.'' Or, there is required a parallel chemical analyses of pure synthetic compounds. Why? Due to the great structural similarity of the mixtures of carbohydrate products of interaction in solution and under MS experimental conditions very frequently these analytes cannot be completely separated even under UPLC conditions.
I should only add, that the largest part of the published MS data on naturally occurring products from microorganism are irreproducible even following the shown experimental conditions, due to facts, mentioned lastly.
The method of mass spectrometry, however, as references [3-5] evidenced empirically, is an exact and absolute method providing exact or absolute 3D structural information and quantitative data at fmol to attomol analytical concentrations.
Attached paper is about:
Magnetic techniques for the isolation and purification of proteins and peptides
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