Contributions of mass spectrometry in the study of nucleic acid‐binding proteins and of nucleic acid–protein interactions

I. Introduction 306 II. Supramolecular Structure Characterization 308A. A Protein Is Used as a “Hook” 309   1. Immuno‐Precipitation 309   2. Tagging with a Poly‐Histidine Sequence 310   3. Direct Analysis of Large Protein Complexes 311   4. Tandem Affinity Purification Tag Procedure 312B. A Nucleic Acid Is Used as a “Hook” or as a “Tracking Label” 314   1. Purification of a Nucleic Acid–Protein Complex in Solution 316   2. Preparative Electrophoretic Mobility Shift Assay 317   3. Surface‐Enhanced Laser Desorption/Ionization 317   4. Surface Plasmon Resonance 319   5. Probing Nucleic Acid–Protein Interactions on Membranes 320 III. Supramolecular Analysis in the Gas Phase 321 IV. Molecular Structure Characterization 324A. Determination of the Assembly Topology 324   1. Non Site‐Directed Cross‐Links 326   2. Native‐State Chemistry 330   3. Site‐Directed Cross‐Links 332B. Regulation of the Complex's Structure and Function 334   1. Protein Phosphorylation 334   2. Modification of the Basic Residues 336   3. Oxidoreduction Balance‐Related Modifications 337 V. Concluding Remarks 339A. Factual Contributions 339B. Perspectives 340 Acknowledgments 341 Abbreviations 341 References 341Nucleic acid–protein (NA–P) interactions play essential roles in a variety of biological processes—gene expression regulation, DNA repair, chromatin structure regulation, transcription regulation, RNA processing, and translation—to cite only a few. Such biological processes involve a broad spectrum of NA–P interactions as well as protein–protein (P–P) interactions. These interactions are dynamic, in terms of the chemical composition of the complexes involved and in terms of their mere existence, which may be restricted to a given cell‐cycle phase. In this review, the contributions of mass spectrometry (MS) to the deciphering of these intricate networked interactions are described along with the numerous applications in which it has proven useful. Such applications include, for example, the identification of the partners involved in NA–P or P–P complexes, the identification of post‐translational modifications that (may) regulate such complexes' activities, or even the precise molecular mapping of the interaction sites in the NA–P complex. From a biological standpoint, we felt that it was worth the reader's time to be as informative as possible about the functional significance of the analytical methods reviewed herein. From a technical standpoint, because mass spectrometry without proper sample preparation would serve no purpose, each application described in this review is detailed by duly emphasizing the sample preparation—whenever this step is considered innovative—that led to significant analytical achievements. © 2003 Wiley Periodicals, Inc., Mass Spec Rev 21:305–348, 2002; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/mas.10036