Shotgun Analysis of Integral Membrane Proteins Facilitated by Elevated Temperature

The beneficial effects on peak selectivity and resolution of conducting liquid chromatography (LC) at elevated temperature (e.g., 30−80 °C) are generally well-known; however, its importance for peptide recovery is not nearly as well recognized. This report demonstrates that μLC analysis of membrane proteomic samples significantly benefits from the application of heat. Enriched membrane and membrane-embedded peptides (the latter obtained by membrane shaving) were analyzed by μLC-tandem mass spectrometry (MS/MS) from 20 to 60 °C using a standard reversed-phase material. Maximal protein and hydrophobic peptide recovery was obtained at 60 °C. The membrane-shaving method employed, a recently optimized version of the high pH/proteinase K protocol, provided significant integral membrane protein enrichment:  98% of identified proteins were predicted to have at least one transmembrane domain (87% to have at least three), and 68% of peptides were predicted to contain transmembrane segments. Analysis of this highly enriched sample at elevated temperature increased protein identifications by 400%, and peptide identifications by 500%, as compared to room-temperature separation. Given that most μLC-MS/MS analyses are currently conducted at room temperature, the findings described herein should be of considerable value for improving the comprehensive study of integral membrane proteins

Shotgun Analysis of Integral Membrane Proteins Facilitated by Elevated Temperature

The beneficial effects on peak selectivity and resolution of conducting liquid chromatography (LC) at elevated temperature (e.g., 30−80 °C) are generally well-known; however, its importance for peptide recovery is not nearly as well recognized. This report demonstrates that μLC analysis of membrane proteomic samples significantly benefits from the application of heat. Enriched membrane and membrane-embedded peptides (the latter obtained by membrane shaving) were analyzed by μLC-tandem mass spectrometry (MS/MS) from 20 to 60 °C using a standard reversed-phase material. Maximal protein and hydrophobic peptide recovery was obtained at 60 °C. The membrane-shaving method employed, a recently optimized version of the high pH/proteinase K protocol, provided significant integral membrane protein enrichment:  98% of identified proteins were predicted to have at least one transmembrane domain (87% to have at least three), and 68% of peptides were predicted to contain transmembrane segments. Analysis of this highly enriched sample at elevated temperature increased protein identifications by 400%, and peptide identifications by 500%, as compared to room-temperature separation. Given that most μLC-MS/MS analyses are currently conducted at room temperature, the findings described herein should be of considerable value for improving the comprehensive study of integral membrane proteins

Shotgun Analysis of Integral Membrane Proteins Facilitated by Elevated Temperature

The beneficial effects on peak selectivity and resolution of conducting liquid chromatography (LC) at elevated temperature (e.g., 30−80 °C) are generally well-known; however, its importance for peptide recovery is not nearly as well recognized. This report demonstrates that μLC analysis of membrane proteomic samples significantly benefits from the application of heat. Enriched membrane and membrane-embedded peptides (the latter obtained by membrane shaving) were analyzed by μLC-tandem mass spectrometry (MS/MS) from 20 to 60 °C using a standard reversed-phase material. Maximal protein and hydrophobic peptide recovery was obtained at 60 °C. The membrane-shaving method employed, a recently optimized version of the high pH/proteinase K protocol, provided significant integral membrane protein enrichment:  98% of identified proteins were predicted to have at least one transmembrane domain (87% to have at least three), and 68% of peptides were predicted to contain transmembrane segments. Analysis of this highly enriched sample at elevated temperature increased protein identifications by 400%, and peptide identifications by 500%, as compared to room-temperature separation. Given that most μLC-MS/MS analyses are currently conducted at room temperature, the findings described herein should be of considerable value for improving the comprehensive study of integral membrane proteins

Shotgun Analysis of Integral Membrane Proteins Facilitated by Elevated Temperature

The beneficial effects on peak selectivity and resolution of conducting liquid chromatography (LC) at elevated temperature (e.g., 30−80 °C) are generally well-known; however, its importance for peptide recovery is not nearly as well recognized. This report demonstrates that μLC analysis of membrane proteomic samples significantly benefits from the application of heat. Enriched membrane and membrane-embedded peptides (the latter obtained by membrane shaving) were analyzed by μLC-tandem mass spectrometry (MS/MS) from 20 to 60 °C using a standard reversed-phase material. Maximal protein and hydrophobic peptide recovery was obtained at 60 °C. The membrane-shaving method employed, a recently optimized version of the high pH/proteinase K protocol, provided significant integral membrane protein enrichment:  98% of identified proteins were predicted to have at least one transmembrane domain (87% to have at least three), and 68% of peptides were predicted to contain transmembrane segments. Analysis of this highly enriched sample at elevated temperature increased protein identifications by 400%, and peptide identifications by 500%, as compared to room-temperature separation. Given that most μLC-MS/MS analyses are currently conducted at room temperature, the findings described herein should be of considerable value for improving the comprehensive study of integral membrane proteins

Shotgun Analysis of Integral Membrane Proteins Facilitated by Elevated Temperature

The beneficial effects on peak selectivity and resolution of conducting liquid chromatography (LC) at elevated temperature (e.g., 30−80 °C) are generally well-known; however, its importance for peptide recovery is not nearly as well recognized. This report demonstrates that μLC analysis of membrane proteomic samples significantly benefits from the application of heat. Enriched membrane and membrane-embedded peptides (the latter obtained by membrane shaving) were analyzed by μLC-tandem mass spectrometry (MS/MS) from 20 to 60 °C using a standard reversed-phase material. Maximal protein and hydrophobic peptide recovery was obtained at 60 °C. The membrane-shaving method employed, a recently optimized version of the high pH/proteinase K protocol, provided significant integral membrane protein enrichment:  98% of identified proteins were predicted to have at least one transmembrane domain (87% to have at least three), and 68% of peptides were predicted to contain transmembrane segments. Analysis of this highly enriched sample at elevated temperature increased protein identifications by 400%, and peptide identifications by 500%, as compared to room-temperature separation. Given that most μLC-MS/MS analyses are currently conducted at room temperature, the findings described herein should be of considerable value for improving the comprehensive study of integral membrane proteins

A Shotgun Proteomic Method for the Identification of Membrane-Embedded Proteins and Peptides

Integral membrane proteins perform crucial cellular functions and are the targets for the majority of pharmaceutical agents. However, the hydrophobic nature of their membrane-embedded domains makes them difficult to work with. Here, we describe a shotgun proteomic method for the high-throughput analysis of the membrane-embedded transmembrane domains of integral membrane proteins which extends the depth of coverage of the membrane proteome

Improved Precision of Proteomic Measurements in Immunoprecipitation Based Purifications Using Relative Quantitation

Mass spectrometry coupled immunoprecipitation (MS-IP) studies are useful in identifying and quantitating potential binding partners of a target protein. However, they are often conducted without appropriate loading controls. Western blots are often used to analyze loading controls, yet there are limitations to their usefulness as analytical tools. One remedy for this is the use of selected reaction monitoring (SRM), where the areas under the curve (AUCs) of peptides from a protein of interest can be normalized to those from the constant regions of the immunoglobulins used for the IP. Using this normalization method, significant changes in relative peptide abundance were observed between samples when there appeared to be an unequal load based on immunoglobulin peptide abundance

Quantitative Improvements in Peptide Recovery at Elevated Chromatographic Temperatures from Microcapillary Liquid Chromatography−Mass Spectrometry Analyses of Brain Using Selected Reaction Monitoring

Elevated chromatographic temperatures are well recognized to provide beneficial analytical effects. Previously, we demonstrated that elevated chromatographic temperature enhances the identification of hydrophobic peptides from enriched membrane samples. Here, we quantitatively assess and compare the recovery of peptide analytes from both simple and complex tryptic peptide matrices using selected reaction monitoring (SRM) mass spectrometry. Our study demonstrates that elevated chromatographic temperature results in significant improvements in the magnitude of peptide recovery for both hydrophilic and hydrophobic peptides from both simple and complex peptide matrices. Importantly, the analytical benefits for quantitative measurements in mouse whole brain matrix are highlighted, suggesting broad utility in the proteomic analyses of complex mammalian tissues. Any improvement in peptide recovery from chromatographic separations translates directly to the apparent sensitivity of downstream mass analysis in microcapillary liquid chromatography-mass spectrometry (μLC−MS) based proteomic applications. Therefore, the incorporation of elevated chromatographic temperatures should result in significant improvements in peptide quantification as well as detection and identification

A Shotgun Proteomic Method for the Identification of Membrane-Embedded Proteins and Peptides

Integral membrane proteins perform crucial cellular functions and are the targets for the majority of pharmaceutical agents. However, the hydrophobic nature of their membrane-embedded domains makes them difficult to work with. Here, we describe a shotgun proteomic method for the high-throughput analysis of the membrane-embedded transmembrane domains of integral membrane proteins which extends the depth of coverage of the membrane proteome

A Shotgun Proteomic Method for the Identification of Membrane-Embedded Proteins and Peptides

Integral membrane proteins perform crucial cellular functions and are the targets for the majority of pharmaceutical agents. However, the hydrophobic nature of their membrane-embedded domains makes them difficult to work with. Here, we describe a shotgun proteomic method for the high-throughput analysis of the membrane-embedded transmembrane domains of integral membrane proteins which extends the depth of coverage of the membrane proteome