Challenges in Separations of Proteins and Small Biomolecules and the Role of Modern Mass Spectroscopy Tools for Solving Them, as Well as Bypassing Them, in Structural Analytical Studies of Complex Biomolecular Mixtures

Abstract

State-of-the-art purification of biomolecules, as well as separation of complex omic mixtures, is crucial for modern biomedical research. Mass spectroscopy (MS) represents a technique that both requires very clean biomedical samples and can substantially assist liquid chromatography (LC) separations, using either LC-MS or LC-MS/MS methods available. Here, a brief overview of the applicability of LC-MS/MS methodology for structural analyses of complex omic mixtures without prior purification of each sample component will be given. When necessary bioinformatic tools are available, these can be carried out quite quickly. However, manual data analysis of such complex mixtures is typically very slow. On the other hand, the need for high-level purity of protein samples for modern biomedical research will be discussed. Often, modification of protein purification protocols is needed, or additional purification steps may be either required or preferred. In the context of mass spectroscopy-related biomedical research, purification of pmol and subpmol amounts of biomedical samples, as well as commercial availability of pmol amounts of purified standards will be discussed.

Enhanced production of recombinant HALT-1 pore-forming toxin using two-step chromatographic procedure

“…Protein impurities with the size of 60-75 kDa were detected in the eluted fractions (Fig. 6A, lanes [5][6][7][8][9][10][11][12][13][14][15][16]. Eluted fractions of rHALT-1 were pooled, and buffer-exchanged in phosphate binding buffer and added to the column containing SP sepharose resins.…”

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2017–2018

“…Two reviews are relevant: "Advances in fractionation and analysis of milk carbohydrates" (130 references) (Nagaraj et al, 2018) and "Challenges in separations of proteins and small biomolecules and the role of modern mass spectroscopy tools for solving them as well as bypassing them, in structural analytical studies of complex biomolecular mixtures". The second half of the review deals with milk sugars (89 references) (Haramija, 2018). A list of the total known human milk sugars has been published (Urashima, Hirabayashi, et al, 2018).…”

Enhanced production of recombinant HALT-1 pore-forming toxin using two-step chromatographic procedure

“…Analytical methods such as mass spectrophotometry and surface plasmon resonance, structural studies such as x-ray crystallography and cryogenic transmission electron microscopy, and the production of therapeutic proteins, all these applications require high purity of protein [12] . As mentioned above, IMAC shall be coupled with the SEC to obtain rHALT-1 without contaminants.…”