TY - JOUR
T1 - Online μSEC2-nRPLC-MS for Improved Sensitivity of Intact Protein Detection of IEF-Separated Nonhuman Primate Cerebrospinal Fluid Proteins
AU - Cline, Erika N.
AU - Alvarez, Carina
AU - Duan, Jiana
AU - Patrie, Steven M.
N1 - Funding Information:
The authors have declared no conflict of interests. This work was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number 1R01GM115739-01A1 and R01GM115739-04S1. Any opinions, findings, conclusions, or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Institutes of Health. This work was also supported by the Multiple Sclerosis Society [PP-1503-04034], The Darrel K. Royal Research Fund for Alzheimer’s Disease [48680-DKR], The Texas Alzheimer’s Research and Care Consortium Investigator Grant Program [354091], and the UT System Neuroscience and Neurotechnology Research Institute [363027]. Funding was also provided by the University of Texas at Dallas, the John L. Roach Scholarship in Biomedical Research, and the Friends of Alzheimer’s Disease Research Award. The authors would like to thank Andrew J. Alpert (polyLC Inc.) for the guidance in the use of PolyHEA columns for SEC and AbbVie Inc. for the kind gift of NHP CSF.
Publisher Copyright:
© 2021 American Chemical Society
PY - 2021/12/21
Y1 - 2021/12/21
N2 - Proteoform-resolved information, obtained by top–down (TD) “intact protein” proteomics, is expected to contribute substantially to the understanding of molecular pathogenic mechanisms and, in turn, identify novel therapeutic and diagnostic targets. However, the robustness of mass spectrometry (MS) analysis of intact proteins in complex biological samples is hindered by the high dynamic range in protein concentration and mass, protein instability, and buffer complexity. Here, we describe an evolutionary step for intact protein investigations through the online implementation of tandem microflow size-exclusion chromatography with nanoflow reversed-phase liquid chromatography and MS (μSEC2-nRPLC-MS). Online serial high-/low-pass SEC filtration overcomes the aforementioned hurdles to intact proteomic analysis through automated sample desalting/cleanup and enrichment of target mass ranges (5–155 kDa) prior to nRPLC-MS. The coupling of μSEC to nRPLC is achieved through a novel injection volume control (IVC) strategy of inserting protein trap columns, pre- and post-μSEC columns, to enable injection of dilute samples in high volumes without loss of sensitivity or resolution. Critical characteristics of the approach are tested via rigorous investigations on samples of varied complexity and chemical background. Application of the platform to cerebrospinal fluid (CSF) prefractionated by OFFGEL isoelectric focusing drastically increases the number of intact mass tags (IMTs) detected within the target mass range (5–30 kDa) in comparison to one-dimensional nRPLC-MS with approximately 100× less CSF than previous OFFGEL studies. Furthermore, the modular design of the μSEC2-nRPLC-MS platform is robust and promises significant flexibility for large-scale TDMS analysis of diverse samples either directly or in concert with other multidimensional fractionation steps.
AB - Proteoform-resolved information, obtained by top–down (TD) “intact protein” proteomics, is expected to contribute substantially to the understanding of molecular pathogenic mechanisms and, in turn, identify novel therapeutic and diagnostic targets. However, the robustness of mass spectrometry (MS) analysis of intact proteins in complex biological samples is hindered by the high dynamic range in protein concentration and mass, protein instability, and buffer complexity. Here, we describe an evolutionary step for intact protein investigations through the online implementation of tandem microflow size-exclusion chromatography with nanoflow reversed-phase liquid chromatography and MS (μSEC2-nRPLC-MS). Online serial high-/low-pass SEC filtration overcomes the aforementioned hurdles to intact proteomic analysis through automated sample desalting/cleanup and enrichment of target mass ranges (5–155 kDa) prior to nRPLC-MS. The coupling of μSEC to nRPLC is achieved through a novel injection volume control (IVC) strategy of inserting protein trap columns, pre- and post-μSEC columns, to enable injection of dilute samples in high volumes without loss of sensitivity or resolution. Critical characteristics of the approach are tested via rigorous investigations on samples of varied complexity and chemical background. Application of the platform to cerebrospinal fluid (CSF) prefractionated by OFFGEL isoelectric focusing drastically increases the number of intact mass tags (IMTs) detected within the target mass range (5–30 kDa) in comparison to one-dimensional nRPLC-MS with approximately 100× less CSF than previous OFFGEL studies. Furthermore, the modular design of the μSEC2-nRPLC-MS platform is robust and promises significant flexibility for large-scale TDMS analysis of diverse samples either directly or in concert with other multidimensional fractionation steps.
UR - http://www.scopus.com/inward/record.url?scp=85121039974&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85121039974&partnerID=8YFLogxK
U2 - 10.1021/acs.analchem.1c00396
DO - 10.1021/acs.analchem.1c00396
M3 - Article
C2 - 34881887
AN - SCOPUS:85121039974
SN - 0003-2700
VL - 93
SP - 16741
EP - 16750
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 50
ER -