Most cited article - PubMed ID 36355445
Reversed-Phase Liquid Chromatography of Peptides for Bottom-Up Proteomics: A Tutorial
Mass spectrometry (MS) has changed our understanding of health, disease, and the environment through untargeted analyses where entire molecular classes are investigated. These techniques generate huge amounts of data which when processed by statistical tools can identify important molecular features or biomarkers. The complexities of these samples are not compatible with direct introduction to the MS system and require a high-resolution separation step, typically low flow liquid chromatography (LC), prior to MS. LC columns that can produce adequate linear velocities at these low flow rates are small in volume making their results susceptible to resolution loss in extra-column volumes. Here, we investigate the implications of the extra-column effects in five LC-MS systems with triple quadrupole and orbitrap mass analyzers. The extra-column volume of these systems in their standard configuration ranged from 26.4 to 78.1 μL which we reduced to 9.57 to 18.7 μL by optimizing the fluidics. The effects of this volume reduction were assessed by studying a hydrolyzed protein sample in a proteomics environment where the intensity of the largest MS peak was improved by 1.8-3.8×. Additionally, the number of molecular features detected in the protein sample improved by up to 7.5×. The relationship between extra-column volumetric variance and flow rate shows that broadening will become much larger for MS detectors at higher flow rates, unlike a traditional small volume UV detector. The methods, applications, and theoretical insights in this work can be used to improve the mass spectrometric results of any LC-MS system.
- Keywords
- LC-MS, band broadening, extra-column effects, instrumentation, liquid chromatography, omics, proteomics,
- Publication type
- Journal Article MeSH
Microbore columns with a 1.0 mm inner diameter (i.d.) have gained popularity in microflow liquid chromatography-mass spectrometry (LC-MS) workflows for exploratory proteomics applications due to their high throughput, robustness, and reproducibility. However, obtaining highly efficient separation using these columns remains unachievable, primarily due to significant radial flow heterogeneity caused by uneven particle packing density across the column cross-section. In this study, we evaluated the integration of a 1.5 mm i.d. column, which offers greater packing uniformity and reduced radial flow dispersion, into a microflow LC-MS setup for bottom-up proteomics analysis. The performance of the 1.5 mm i.d. column was compared with that of the 1.0 mm i.d. column using protein samples of varying complexity. The results demonstrate that 1.5 mm i.d. columns provide superior chromatographic separation and better compatibility with conventional-flow LC systems, yielding higher reproducibility and comparable protein and peptide identifications to the 1.0 mm i.d. columns at higher sample amounts. These findings suggest that 1.5 mm i.d. columns could be a suitable alternative to 1.0 mm i.d. columns for microflow LC-MS/MS proteomic analysis, particularly in laboratories with only conventional-flow LC systems.
- Publication type
- Journal Article MeSH
Over 400 different types of post-translational modifications (PTMs) have been reported and over 200 various types of PTMs have been discovered using mass spectrometry (MS)-based proteomics. MS-based proteomics has proven to be a powerful method capable of global PTM mapping with the identification of modified proteins/peptides, the localization of PTM sites and PTM quantitation. PTMs play regulatory roles in protein functions, activities and interactions in various heart related diseases, such as ischemia/reperfusion injury, cardiomyopathy and heart failure. The recognition of PTMs that are specific to cardiovascular pathology and the clarification of the mechanisms underlying these PTMs at molecular levels are crucial for discovery of novel biomarkers and application in a clinical setting. With sensitive MS instrumentation and novel biostatistical methods for precise processing of the data, low-abundance PTMs can be successfully detected and the beneficial or unfavorable effects of specific PTMs on cardiac function can be determined. Moreover, computational proteomic strategies that can predict PTM sites based on MS data have gained an increasing interest and can contribute to characterization of PTM profiles in cardiovascular disorders. More recently, machine learning- and deep learning-based methods have been employed to predict the locations of PTMs and explore PTM crosstalk. In this review article, the types of PTMs are briefly overviewed, approaches for PTM identification/quantitation in MS-based proteomics are discussed and recently published proteomic studies on PTMs associated with cardiovascular diseases are included.
- Keywords
- MS‐based proteomics, cardiovascular disease, post‐translational modifications, proteins,
- MeSH
- Phosphorylation MeSH
- Mass Spectrometry methods MeSH
- Cardiovascular Diseases * metabolism genetics pathology MeSH
- Humans MeSH
- Protein Processing, Post-Translational * MeSH
- Proteomics * methods MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
Elevating the column temperature is an effective strategy for improving the chromatographic separation of peptides. However, high temperatures induce artificial modifications that compromise the quality of the peptide analysis. Here, we present a novel high-temperature LC-MS method that retains the benefits of a high column temperature while significantly reducing peptide modification and degradation during reversed-phase liquid chromatography. Our approach leverages a short inline trap column maintained at a near-ambient temperature installed upstream of a separation column. The retentivity and dimensions of the trap column were optimized to shorten the residence time of peptides in the heated separation column without compromising the separation performance. This easy-to-implement approach increased peak capacity by 1.4-fold within a 110 min peptide mapping of trastuzumab and provided 10% more peptide identifications in exploratory LC-MS proteomic analyses compared with analyses conducted at 30 °C while maintaining the extent of modifications close to the background level. In the peptide mapping of biopharmaceuticals, where in-column modifications can falsely elevate the levels of some critical quality attributes, the method reduced temperature-related artifacts by 66% for N-terminal pyroGlu and 63% for oxidized Met compared to direct injection at 60 °C, thus improving reliability in quality control of protein drugs. Our findings represent a promising advancement in LC-MS methodology, providing researchers and industry professionals with a valuable tool for improving the chromatographic separation of peptides while significantly reducing the unwanted modifications.
- MeSH
- Biological Products analysis chemistry MeSH
- Chromatography, Liquid methods MeSH
- Mass Spectrometry MeSH
- Liquid Chromatography-Mass Spectrometry MeSH
- Peptides analysis chemistry MeSH
- Proteins analysis isolation & purification chemistry MeSH
- Proteomics * methods MeSH
- Quality Control * MeSH
- Trastuzumab chemistry analysis MeSH
- Hot Temperature MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Biological Products MeSH
- Peptides MeSH
- Proteins MeSH
- Trastuzumab MeSH
Sample preparation involving the cleavage of proteins into peptides is the first critical step for successful bottom-up proteomics and protein analyses. Time- and labor-intensiveness are among the bottlenecks of the commonly used methods for protein sample preparation. Here, we report a fast online method for postinjection acid cleavage of proteins directly in the mobile phase typically used for LC-MS analyses in proteomics. The chemical cleavage is achieved in 0.1% formic acid within 35 s in a capillary heated to 195 °C installed upstream of the analytical column, enabling the generated peptides to be separated. The peptides generated by the optimized method covered the entire sequence except for one amino acid of trastuzumab used for the method development. The qualitative results are extraordinarily stable, even over a long period of time. Moreover, the method is also suitable for accurate and repeatable quantification. The procedure requires only one manual step, significantly decreasing sample transfer losses. To demonstrate its practical utility, we tested the method for the fast detection of ricin. Ricin can be unambiguously identified from an injection of 10 ng, and the results can be obtained within 7-8 min after receiving a suspicious sample. Because no sophisticated accessories and no additional reagents are needed, the method can be seamlessly transferred to any laboratory for high-throughput proteomic workflows.
