Characterisation of a new online nanoLC-CZE-MS platform and application for the glycosylation profiling of alpha-1-acid glycoprotein

The ever-increasing complexity of biological samples to be analysed by mass spectrometry has led to the necessity of sophisticated separation techniques, including multidimensional separation. Despite a high degree of orthogonality, the coupling of liquid chromatography (LC) and capillary zone electrophoresis (CZE) has not gained notable attention in research. Here, we present a heart-cut nanoLC-CZE-ESI-MS platform to analyse intact proteins. NanoLC and CZE-MS are coupled using a four-port valve with an internal nanoliter loop. NanoLC and CZE-MS conditions were optimised independently to find ideal conditions for the combined setup. The valve setup enables an ideal transfer efficiency between the dimensions while maintaining good separation conditions in both dimensions. Due to the higher loadability, the nanoLC-CZE-MS setup exhibits a 280-fold increased concentration sensitivity compared to CZE-MS. The platform was used to characterise intact human alpha-1-acid glycoprotein (AGP), an extremely heterogeneous N-glycosylated protein. With the nanoLC-CZE-MS approach, 368 glycoforms can be assigned at a concentration of 50 μg/mL as opposed to the assignment of only 186 glycoforms from 1 mg/mL by CZE-MS. Additionally, we demonstrate that glycosylation profiling is accessible for dried blood spot analysis (25 μg/mL AGP spiked), indicating the general applicability of our setup to biological matrices. The combination of high sensitivity and orthogonal selectivity in both dimensions makes the here-presented nanoLC-CZE-MS approach capable of detailed characterisation of intact proteins and their proteoforms from complex biological samples and in physiologically relevant concentrations. Graphical abstract

Pulsed Nanoelectrospray Ionization Boosts Ion Signal in Whole Protein Mass Spectrometry

Electrospray ionisation (ESI) is renowned for its ability to ionise intact proteins for sensitive detection by mass spectrometry (MS). However, the use of a conventional direct current ESI voltage can result in the formation of relatively large initial droplet sizes, which can limit efficient ion desolvation and sensitivity. Here, pulsed nanoESI (nESI) MS using nanoscale emitters with inner diameters of ~250 nm is reported. In this approach, the nESI voltage is rapidly pulsed from 0 to ~1.5 kV with sub-nanosecond rise times, duty cycles from 10 to 90%, and repetition rates of 10 to 350 kHz. Using pulsed nESI, the performance of MS for the detection of intact proteins can be improved in terms of increased ion abundances and decreased noise. The absolute ion abundances and signal-to-noise levels of protonated ubiquitin, cytochrome C, myoglobin, and carbonic anhydrase II formed from standard denaturing solutions can be increased by up to 82% and 154% using an optimal repetition rate of ~200 kHz compared to conventional nESI-MS. Applying pulsed nESI-MS to a mixture of four proteins resulted in the signal for each protein increasing by up to 184% compared to the more conventional nESI-MS. For smaller ions (≤1032 m/z), the signal can also be increased by the use of high repetition rates (200–250 kHz), which is consistent with the enhanced performance depending more on general factors associated with the ESI process (e.g., smaller initial droplet sizes and reduced Coulombic repulsion in the spray plume) rather than analyte-specific effects (e.g., electrophoretic mobility). The enhanced sensitivity of pulsed nESI is anticipated to be beneficial for many different types of tandem mass spectrometry measurements.

FLASHIda: Intelligent data acquisition for top-down proteomics that doubles proteoform level identification count

Top-down proteomics (TDP) has gained a lot of interest in biomedical application for detailed analysis and structural characterization of proteoforms. Data-dependent acquisition (DDA) of intact proteins is non-trivial due to the diversity and complex signal of proteoforms. Dedicated acquisition methods thus have the potential to greatly improve TDP. We present FLASHIda, an intelligent online data acquisition algorithm for TDP that ensures the real-time selection of high-quality precursors of diverse proteoforms. FLASHIda combines fast charge deconvolution algorithms and machine learning-based quality assessment for optimal precursor selection. In analysis in E. coli lysates, FLASHIda increased the number of unique proteoform level identifications from 800 to 1,500, or generated a near-identical number of identifications in ⅓ of instrument time when compared to standard DDA mode. Furthermore, FLASHIda enabled sensitive mapping of post translational modifications and detection of chemical adducts. As an extension module to the instrument, FLASHIda can be readily adopted for TDP studies of complex samples to enhance proteoform identification rates.

Unveiling the Metabolic Effects of Glycomacropeptide

For many years, the main nitrogen source for patients with phenylketonuria (PKU) was phenylalanine-free amino acid supplements. Recently, casein glycomacropeptide (GMP) supplements have been prescribed due to its functional and sensorial properties. Nevertheless, many doubts still persist about the metabolic effects of GMP compared to free amino acids (fAA) and intact proteins such as casein (CAS). We endeavour to compare, in rats, the metabolic effects of different nitrogen sources. Twenty-four male Wistar rats were fed equal energy density diets plus CAS (control, n = 8), fAA (n = 8) or GMP (n = 8) for 8 weeks. Food, liquid intake and body weight were measured weekly. Blood biochemical parameters and markers of glycidic metabolism were assessed. Glucagon-like peptide-1 (GLP-1) was analysed by ELISA and immunohistochemistry. Food intake was higher in rats fed CAS compared to fAA or GMP throughout the treatment period. Fluid intake was similar between rats fed fAA and GMP. Body weight was systematically lower in rats fed fAA and GMP compared to those fed CAS, and still, from week 4 onwards, there were differences between fAA and GMP. None of the treatments appeared to induce consistent changes in glycaemia, while insulin levels were significantly higher in GMP. Likewise, the production of GLP-1 was higher in rats fed GMP when compared to fAA. Decreased urea, total protein and triglycerides were seen both in fAA and GMP related to CAS. GMP also reduced albumin and triglycerides in comparison to CAS and fAA, respectively. The chronic consumption of the diets triggers different metabolic responses which may provide clues to further study potential underlying mechanisms.

A Laconic Review on Liquid Chromatography Mass Spectrometry (LC-MS) Based Proteomics Technology in Drug Discovery

Liquid chromatography mass spectrometry is a powerful technique which is used for the new product development, manufacturing, and to the control the stability or drug abuse. These techniques were commonly preferred in pharmacokinetics studies in pharmaceutical products. The principle involved in the LC-MS technique was justified in our study to understand the basic fundamentals of LC-MS. Also, it is used in combination with HPLC for chemical analysis. Many applications and the future prospects have been highlighted regarding the use for LC-MS in analytical chemistry. It is basically depends on the metabolites which are collected in this technique, furthermore the chemicals are analyzed according to the nature of suitability of LC-MS. This technique was helpful in the analysis of protein components identified in terms of pharmacovigilance, organic/inorganic hybrid nanoflowering. Also, our study highlights the techniques involved in proteomics. In LC-MS-based proteomics, complex mixtures of proteins are first subjected to enzymatic cleavage, then the resulting peptide products are analyzed using a mass spectrometer; this is in contrast to "top-down" proteomics, which deals with intact proteins and is limited to single protein mixtures. So, this review aims to highlight the basic introduction, and principle involved in liquid chromatography-mass spectrometry (LC-MS). Also, the advantages, or application of LC-MS were studied. Most importantly the LC-MS based proteomics, and the future aspects of LC-MS technology were studied in this review.