Developing a Multiplexed Quantitative Cross-Linking Mass Spectrometry Platform for Comparative Structural Analysis of Protein Complexes.
Cross-linking mass spectrometry (XL-MS) represents a recently popularized hybrid methodology for defining protein-protein interactions (PPIs) and analyzing structures of large protein assemblies. In particular, XL-MS strategies have been demonstrated to be effective in elucidating molecular details of PPIs at the peptide resolution, providing a complementary set of structural data that can be utilized to refine existing complex structures or direct de novo modeling of unknown protein structures. To study structural and interaction dynamics of protein complexes, quantitative cross-linking mass spectrometry (QXL-MS) strategies based on isotope-labeled cross-linkers have been developed. Although successful, these approaches are mostly limited to pairwise comparisons. In order to establish a robust workflow enabling comparative analysis of multiple cross-linked samples simultaneously, we have developed a multiplexed QXL-MS strategy, namely, QMIX (Quantitation of Multiplexed, Isobaric-labeled cross (X)-linked peptides) by integrating MS-cleavable cross-linkers with isobaric labeling reagents. This study has established a new analytical platform for quantitative analysis of cross-linked peptides, which can be directly applied for multiplexed comparisons of the conformational dynamics of protein complexes and PPIs at the proteome scale in future studies.
Department of Physiology & Biophysics, University of California, Irvine , Irvine, California 92697, United States.
Clinton
C
Yu
Department of Physiology & Biophysics, University of California, Irvine , Irvine, California 92697, United States.
Alexander
A
Huszagh
Thermo Fisher , 355 River Oaks Parkway, San Jose, California 95134, United States.
Rosa
R
Viner
Department of Chemistry, University of California, Irvine , Irvine, California 92697, United States.
Eric J
EJ
Novitsky
Department of Chemistry, University of California, Irvine , Irvine, California 92697, United States.
Scott D
SD
Rychnovsky
Department of Physiology & Biophysics, University of California, Irvine , Irvine, California 92697, United States.
Lan
L
Huang
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000737:chemistry*; D045304:Cytochromes c / Q000737:chemistry / Q000378:metabolism; D007553:Isotope Labeling; D010455:Peptides / Q000032:analysis; D054730:Protein Interaction Domains and Motifs; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D013454:Sulfoxides / Q000737:chemistry; D053719:Tandem Mass Spectrometry
D003432:Cross-Linking Reagents; D010455:Peptides; D011506:Proteins; D013454:Sulfoxides; D045304:Cytochromes c