Proteomic tools for quantitation by mass spectrometry

  I. Introduction 182  II. Relative Quantitation 184A.  2‐D Gel Electrophoresis 184B.  Metabolic Isotopic Labeling 185    1.   15 N 185    2.   13 C Enrichment and Depletion 185    3.  Select Isotopic Amino Acid Incorporation 186C.  Chemical Labeling 186    1.  Labeling During Proteolysis:  18 O 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 189a.  Nicotinyl‐ N ‐hyxroxysuccinimide 189b.  Acylation 189c.  Amidination‐quantitation using enhanced signal tags (QUEST) 190d.  C‐terminusu labeling 190D.  Differential Mass Mapping 190 III. Absolute Quantitation 191  IV. Choice of Instrumentation 191   V. Overview, Discussion, and Future Directions 191 References 192Techniques 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