Enhanced Payload Localization in Antibody-Drug Conjugates Using a Middle-Down Mass Spectrometry Approach with Proton Transfer Charge Reduction.

Antibody-drug conjugates (ADCs) represent a novel class of immunoconjugates with growing therapeutic relevance, since they combine the efficacy of cytotoxic drugs with the specificity of antibodies. However, by design, ADCs introduce structural features into the monoclonal antibody scaffold that complicate their analysis. Payload attachment to cysteine or lysine residues can often result in product heterogeneity, regarding both the number of attached drug molecules and their conjugation site, necessitating the use of state-of-the-art MS instrumentation to elucidate their complexity.

Taylor-Aris Dispersion-Assisted Mass Spectrometry for the Analysis of Native Proteins.

As has recently been shown, Taylor-Aris dispersion-assisted mass spectrometry (TADA-MS) can offer direct injection MS determinations in fields where the targets of the analyses are large molecules present in a matrix that would otherwise cause serious interferences. In the present study, we demonstrated the exceptional utility of TADA-MS in native protein analysis: (i) a dramatic improvement in detection sensitivity was found due to its ability to strongly reduce matrix interferences, (ii) more "native-like" conditions can be used during analyses, (iii) the direct injection of non-MS-compatible matrices is allowed into MS, and (iv) a considerable simplification and economization of the workflow is ensured. We investigated the behavior of different types of proteins and protein complexes present under native conditions, demonstrating the unambiguous benefits and simplicity of the method.

Direct Injection Electrospray Ionization Mass Spectrometry (ESI-MS) Analysis of Proteins with High Matrix Content:Utilizing Taylor-Aris Dispersion.

This is the first work demonstrating the utility of the Taylor-Aris (TA) dispersion in avoiding serious interference issues commonly occurring in the electrospray ionization-mass spectrometric (ESI-MS) determination of therapeutic protein pharmaceuticals undergoing no pre-separation or sample purification. It was also pointed out that the TA dispersion conditions and its analytical utilization for proteomics can be easily accomplished in a commercial CE-MS instrument. In the proposed Taylor-Aris dispersion-assisted mass spectrometry (TADA-MS) analysis 0.

CZE-MS peptide mapping: To desalt or not to desalt?

Background: In "shotgun" approaches involving high-performance liquid chromatography or capillary zone electrophoresis (CZE), matrix removal prior to sample analysis is considered as an indispensable tool. Despite the fact that CZE offers a high tolerance towards salts, most publications reported on the use of desalting. There seems to be no clear consensus on the utilization of desalting in the CZE-MS community, most probably due to the absence of works addressing the comparison of desalted and non-desalted digests.

Recombinant Expression in System of Three Potent Kv1.3 Channel Blockers: Vm24, Anuroctoxin, and Ts6.

The Kv1.3 channel has become a therapeutic target for the treatment of various diseases. Several Kv1.

Determination of human insulin and its six therapeutic analogues by capillary electrophoresis - mass spectrometry.

In this work, human insulin and its 6 analogues were separated and determined using CZE-MS. Three different capillaries (bare fused silica, successive multiple ionic-polymer layer (SMIL) and static linear polyacrylamide (LPA) coated) were compared based on their separation performances in their optimal operating conditions. Coated capillaries demonstrated slightly better separation of the components, although some components showed wide, distorted peaks.

Microfluidic Immobilized Enzymatic Reactors for Proteomic Analyses-Recent Developments and Trends (2017-2021).

Given the strong interdisciplinary nature of microfluidic immobilized enzyme reactor (μ-IMER) technology, several branches of science contribute to its successful implementation. A combination of physical, chemical knowledge and engineering skills is often required. The development and application of μ-IMERs in the proteomic community are experiencing increasing importance due to their attractive features of enzyme reusability, shorter digestion times, the ability to handle minute volumes of sample and the prospect of on-line integration into analytical workflows.

Development of an In-Line Enzyme Reactor Integrated into a Capillary Electrophoresis System.

The goal of this paper was to develop an in-line immobilized enzyme reactor (IMER) integrated into a capillary electrophoresis platform. In our research, we created the IMER by adsorbing trypsin onto the inner surface of a capillary in a short section. Enzyme immobilization was possible due to the electrostatic attraction between the oppositely charged fused silica capillary surface and trypsin.

Study of the geometry of open channels in a layer-bed-type microfluidic immobilized enzyme reactor.

This paper aims at studying open channel geometries in a layer-bed-type immobilized enzyme reactor with computer-aided simulations. The main properties of these reactors are their simple channel pattern, simple immobilization procedure, regenerability, and disposability; all these features make these devices one of the simplest yet efficient enzymatic microreactors. The high surface-to-volume ratio of the reactor was achieved using narrow (25-75 μm wide) channels.

Determination of artemisinin and its analogs in Artemisia annua extracts by capillary electrophoresis - Mass spectrometry.

The applicability of micellar electrokinetic capillary chromatography (MEKC) with mass spectrometric detection for the determination of artemisinin and its analogs (e.g. ascaridole, artemisia ketone, casticin, deoxyartemisinin, arteannuic acid, artemetin, dihydroartemisinic acid) was studied.

Analysis of fumonisin mycotoxins with capillary electrophoresis - mass spectrometry.

This paper demonstrates the development of an analytical method based on CE coupled to ESI-MS for the identification and quantification of fumonisin mycotoxins. Separation and detection parameters (pH of background electrolyte (BGE), organic modifier content, sheath liquid (SL) composition, MS mode and nebuliser pressure) were optimised. Ammonium formate/ammonia (pH = 9.

Fabrication of immobilized enzyme reactors with pillar arrays into polydimethylsiloxane microchip.

This paper demonstrates the design, efficiency and applicability of a simple and inexpensive microfluidic immobilized enzymatic reactor (IMER) for rapid protein digestion. The high surface-to-volume ratio (S/V) of the reactor was achieved by forming pillars in the channel. It was found that pillar arrays including dimensions of 40 μm × 40 μm as pillar diameter and interpillar distance can provide both relatively high S/V and flow rate in the PDMS chip, the fabrication of which was performed by means of soft lithography using average research laboratory infrastructure.

The application of a microfluidic reactor including spontaneously adsorbed trypsin for rapid protein digestion of human tear samples.

Purpose: The application of a newly developed microfluidic immobilized enzymatic reactor (IMER) designed to accelerate protein digestion in clinical samples is presented.

Experimental Design: The IMER contains trypsin adsorbed on the porous surface of a PDMS microfluidic chip. Human tear with its relatively low volume and high protein content is collected and used for testing the digestion efficiency of the IMER.