Replacing immunoassays with tryptic digestion-peptide immunoaffinity enrichment and LC-MS/MS.
For decades, immunoassays have provided the framework for protein biomarker studies in clinical medicine and in therapeutic monitoring for drug development. At the same time, investigators have uncovered many issues that make immunoassays unreliable in many human serum and plasma samples. LC-MS/MS after tryptic digestion of proteins is potentially an attractive solution, but the sensitivity of the method is not sufficient to measure many important low-abundance proteins directly. The use of antipeptide antibodies to immunoenrich peptides of interest can improve the sensitivity of the approach, greatly simplify the matrix enabling shortened chromatographic runs, and facilitate the multiplexed quantification of analytes, which could reduce the costs of quantitative protein measurements in complex specimens. We provide an overview of the method and the steps needed to develop an assay. In addition, we review the efforts to make this method generally more applicable.
Department of Laboratory Medicine, University of Washington, Seattle, WA 98195-7110, USA.
Jessica O
JO
Becker
Andrew N
AN
Hoofnagle
D000906:Antibodies / Q000276:immunology; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D007118:Immunoassay; D010455:Peptides / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D011506:Proteins / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000378:metabolism*
D000906:Antibodies; D010455:Peptides; D011506:Proteins; D014357:Trypsin