Proteomic tools for quantitation by mass spectrometry

Abstract

I. Introduction 182 II. Relative Quantitation 184 A. 2‐D Gel Electrophoresis 184 B. Metabolic Isotopic Labeling 185 1. ¹⁵N 185 2. ¹³C Enrichment and Depletion 185 3. Select Isotopic Amino Acid Incorporation 186 C. Chemical Labeling 186 1. Labeling During Proteolysis: ¹⁸O Incorporation During Enzymatic Cleavage 186 2. Isotopic Tags—Isotope‐Codes Affinity Tag Reagents (ICAT) 187 3. Isotopic Tags—Acid‐Labile Isotope‐Coded Extractants (ALICE) 188 4. Lysine‐Specific Labeling 189 5. Phosphoserine‐ and Phosphothreonine‐Specific Labeling 189 6. N‐Terminus Labeling 189 a. Nicotinyl‐N‐hyxroxysuccinimide 189 b. Acylation 189 c. Amidination‐quantitation using enhanced signal tags (QUEST) 190 d. C‐terminusu labeling 190 D. Differential Mass Mapping 190 III. Absolute Quantitation 191 IV. Choice of Instrumentation 191 V. Overview, Discussion, and Future Directions 191 References 192 Techniques for the quantitation of proteins and peptides by mass spectrometry (MS) are reviewed. A range of labeling processes is discussed, including metabolic, enzymatic, and chemical labeling, and techniques that can be employed for comparative and absolute quantitation are presented. Advantages and drawbacks of the techniques are discussed, and suggestions for the appropriate uses of the methodologies are explained. Overall, the metabolic incorporation of isotopic labels provides the most accurate labeling strategy, and is most useful when an internal standard for comparative quantitation is needed. However, that technique is limited to research that uses cultured cells. © 2003 Wiley Periodicals, Inc., Mass Spec Rev 22:182–194, 2003; Published online in Wiley Interscience ( www.interscience.wiley.com). DOI 10.1002/mas.10048