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Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry
Published on: July 31, 2011
Identification of Antibody-Cognate Antigens via a High-Throughput, Affinity Chromatography-Based, Shotgun
Nicholas A Shortreed1, Kiran K Mangalaparthi2, Benjamin J Madden3
1Mayo Clinic Graduate School of Biomedical Sciences & Department of Immunology, Mayo Clinic, Rochester, MN, USA.
Since they were first described in the late nineteenth century, antibodies have become widely recognized as being able to serve mechanistic functions in both "protective," as well as "pathogenic" adaptive immune responses. Due to their critical roles in a wide range of disease states, there has been significant interest in the fields of immunology and medicine in identifying individual antibody-cognate antigens. However, numerous technical and logistical challenges associated with older cognate antigen identification strategies limited their widespread utilization in these fields. To meet this critical need, we recently developed an optimized, high-throughput, affinity chromatography-based, shotgun immunoproteomics pipeline which we have previously demonstrated resolves many of the identified weaknesses of past methodologies. This chapter provides a comprehensive protocol for each step of this shotgun immunoproteomics pipeline, from input protein sample preparation through data analysis and antigen identification. This high-throughput methodology for antibody-cognate antigen identification is both a user-friendly and cost-effective procedure which could potentially facilitate novel discoveries in basic, translational, and clinical research settings.
Since they were first described in the late nineteenth century, antibodies have become widely recognized as being able to serve mechanistic functions in both "protective," as well as "pathogenic" adaptive immune responses. Due to their critical roles in a wide range of disease states, there has been significant interest in the fields of immunology and medicine in identifying individual antibody-cognate antigens. However, numerous technical and logistical challenges associated with older cognate antigen identification strategies limited their widespread utilization in these fields. To meet this critical need, we recently developed an optimized, high-throughput, affinity chromatography-based, shotgun immunoproteomics pipeline which we have previously demonstrated resolves many of the identified weaknesses of past methodologies. This chapter provides a comprehensive protocol for each step of this shotgun immunoproteomics pipeline, from input protein sample preparation through data analysis and antigen identification. This high-throughput methodology for antibody-cognate antigen identification is both a user-friendly and cost-effective procedure which could potentially facilitate novel discoveries in basic, translational, and clinical research settings.
