More Like this

1. Recent advances (2019–2021) of capillary electrophoresis‐mass spectrometry for multilevel proteomics

   https://doi.org/10.1002/mas.21714  

   Chen, Daoyang; McCool, Elijah N.; Yang, Zhichang; Shen, Xiaojing; Lubeckyj, Rachele A.; Xu, Tian; Wang, Qianjie; Sun, Liangliang (June 2021, Mass Spectrometry Reviews)

   Multilevel proteomics aims to delineate proteins at the peptide (bottom-up proteomics), proteoform (top-down proteomics), and protein complex (native proteomics) levels. Capillary electrophoresis-mass spectrometry (CE-MS) can achieve highly efficient separation and highly sensitive detection of complex mixtures of peptides, proteoforms, and even protein complexes because of its substantial technical progress. CE-MS has become a valuable alternative to the routinely used liquid chromatography-mass spectrometry for multilevel proteomics. This review summarizes the most recent (2019-2021) advances of CE-MS for multilevel proteomics regarding technological progress and biological applications. We also provide brief perspectives on CE-MS for multilevel proteomics at the end, highlighting some future directions and potential challenges. 

   more » « less
2. Mass spectrometry-based technologies for probing the 3D world of plant proteins

   https://doi.org/10.1093/plphys/kiac039  

   Blackburn, Matthew R; Minkoff, Benjamin B; Sussman, Michael R (February 2022, Plant Physiology)

   Abstract Over the past two decades, mass spectrometric (MS)-based proteomics technologies have facilitated the study of signaling pathways throughout biology. Nowhere is this needed more than in plants, where an evolutionary history of genome duplications has resulted in large gene families involved in posttranslational modifications and regulatory pathways. For example, at least 5% of the Arabidopsis thaliana genome (ca. 1,200 genes) encodes protein kinases and protein phosphatases that regulate nearly all aspects of plant growth and development. MS-based technologies that quantify covalent changes in the side-chain of amino acids are critically important, but they only address one piece of the puzzle. A more crucially important mechanistic question is how noncovalent interactions—which are more difficult to study—dynamically regulate the proteome’s 3D structure. The advent of improvements in protein 3D technologies such as cryo-electron microscopy, nuclear magnetic resonance, and X-ray crystallography has allowed considerable progress to be made at this level, but these methods are typically limited to analyzing proteins, which can be expressed and purified in milligram quantities. Newly emerging MS-based technologies have recently been developed for studying the 3D structure of proteins. Importantly, these methods do not require protein samples to be purified and require smaller amounts of sample, opening the wider proteome for structural analysis in complex mixtures, crude lysates, and even in intact cells. These MS-based methods include covalent labeling, crosslinking, thermal proteome profiling, and limited proteolysis, all of which can be leveraged by established MS workflows, as well as newly emerging methods capable of analyzing intact macromolecules and the complexes they form. In this review, we discuss these recent innovations in MS-based “structural” proteomics to provide readers with an understanding of the opportunities they offer and the remaining challenges for understanding the molecular underpinnings of plant structure and function. 

   more » « less
3. Membrane Ultrafiltration-Based Sample Preparation Method and Sheath-Flow CZE-MS/MS for Top-Down Proteomics

   Zhichang Yang, Liangliang Sun (June 2022, Methods in molecular biology)

   Mass spectrometry (MS)-based denaturing top-down proteomics (dTDP) identify proteoforms without pretreatment of enzyme proteolysis. A universal sample preparation method that can efficiently extract protein, reduce sample loss, maintain protein solubility, and be compatible with following up liquid-phase separation, MS, and tandem MS (MS/MS) is vital for large-scale proteoform characterization. Membrane ultrafiltration (MU) was employed here for buffer exchange to efficiently remove the sodium dodecyl sulfate (SDS) detergent in protein samples used for protein extraction and solubilization, followed by capillary zone electrophoresis (CZE)-MS/MS analysis. The MU method showed good protein recovery, minimum protein bias, and nice compatibility with CZE-MS/MS. Single-shot CZE-MS/MS analysis of an Escherichia coli sample prepared by the MU method identified over 800 proteoforms. 

   more » « less
4. SPIN enables high throughput species identification of archaeological bone by proteomics

   https://doi.org/10.1038/s41467-022-30097-x  

   Rüther, Patrick Leopold; Husic, Immanuel Mirnes; Bangsgaard, Pernille; Gregersen, Kristian Murphy; Pantmann, Pernille; Carvalho, Milena; Godinho, Ricardo Miguel; Friedl, Lukas; Cascalheira, João; Taurozzi, Alberto John; et al (December 2022, Nature Communications)

   Abstract Species determination based on genetic evidence is an indispensable tool in archaeology, forensics, ecology, and food authentication. Most available analytical approaches involve compromises with regard to the number of detectable species, high cost due to low throughput, or a labor-intensive manual process. Here, we introduce “Species by Proteome INvestigation” (SPIN), a shotgun proteomics workflow for analyzing archaeological bone capable of querying over 150 mammalian species by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Rapid peptide chromatography and data-independent acquisition (DIA) with throughput of 200 samples per day reduce expensive MS time, whereas streamlined sample preparation and automated data interpretation save labor costs. We confirm the successful classification of known reference bones, including domestic species and great apes, beyond the taxonomic resolution of the conventional peptide mass fingerprinting (PMF)-based Zooarchaeology by Mass Spectrometry (ZooMS) method. In a blinded study of degraded Iron-Age material from Scandinavia, SPIN produces reproducible results between replicates, which are consistent with morphological analysis. Finally, we demonstrate the high throughput capabilities of the method in a high-degradation context by analyzing more than two hundred Middle and Upper Palaeolithic bones from Southern European sites with late Neanderthal occupation. While this initial study is focused on modern and archaeological mammalian bone, SPIN will be open and expandable to other biological tissues and taxa. 

   more » « less
5. Bioinformatics Methods for Mass Spectrometry-Based Proteomics Data Analysis

   https://doi.org/10.3390/ijms21082873  

   Chen, Chen; Hou, Jie; Tanner, John J.; Cheng, Jianlin (April 2020, International Journal of Molecular Sciences)

   Recent advances in mass spectrometry (MS)-based proteomics have enabled tremendous progress in the understanding of cellular mechanisms, disease progression, and the relationship between genotype and phenotype. Though many popular bioinformatics methods in proteomics are derived from other omics studies, novel analysis strategies are required to deal with the unique characteristics of proteomics data. In this review, we discuss the current developments in the bioinformatics methods used in proteomics and how they facilitate the mechanistic understanding of biological processes. We first introduce bioinformatics software and tools designed for mass spectrometry-based protein identification and quantification, and then we review the different statistical and machine learning methods that have been developed to perform comprehensive analysis in proteomics studies. We conclude with a discussion of how quantitative protein data can be used to reconstruct protein interactions and signaling networks. 

   more » « less