Rapid and sensitive identification of epitope-containing peptides by direct matrix-assisted laser desorption/ionization tandem mass spectrometry of peptides affinity-bound to antibody beads.
A method has been developed for rapid and sensitive identification of epitope-containing peptides, based on direct MALDI-MS/MS analysis of epitope-containing peptides affinity bound to affinity beads. This technique provides sequence information of the epitope that allows unambiguous identification of the epitope either by database searching or de novo sequencing. With MALDI-MS, affinity beads with bound peptides can be placed directly on the MALDI target and analyzed. Coupling a MALDI source to an orthogonal injection quadrupole time-of-flight (QqTOF) mass spectrometer allows direct sequencing of the bound peptides. In contrast to ESI-MS/MS, elution of the affinity-bound peptides followed by additional concentration and purification steps is not required, thus reducing the potential for sample loss. Direct mass spectrometric sequencing of affinity-bound peptides eliminates the need for chemical or enzymatic sequencing. Other advantages of this direct MALDI-MS/MS analysis of epitope-containing peptides bound to the affinity beads include its sensitivity (femtomole levels) and speed. In addition, direct analysis of peptides on affinity beads does not adversely affect the high mass accuracy of a QqTOF, and database searching can be performed on the MS/MS spectra obtained. In proof-of-principle experiments, this method has been demonstrated on beads containing immobilized antibodies against phosphotyrosine, the c-myc epitope tag, as well as immobilized avidin. Furthermore, de novo sequencing of epitope-containing peptides is demonstrated. The first application of this method was with anti-FLAG-tag affinity beads, where direct MALDI MS/MS was used to determine an unexpected enzymatic cleavage site on a growth factor protein.
Raska
Christina S
CS
Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Parker
Carol E
CE
Sunnarborg
Susan W
SW
Pope
R Marshall
RM
Lee
David C
DC
Glish
Gary L
GL
Borchers
Christoph H
CH
D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology*; D045163:Antibodies, Phospho-Specific / Q000276:immunology; D000918:Antibody Specificity; D001360:Avidin / Q000378:metabolism; D001710:Biotin / Q000378:metabolism; D000939:Epitopes / Q000032:analysis* / Q000737:chemistry / Q000276:immunology*; D008863:Microspheres; D010455:Peptides / Q000032:analysis* / Q000737:chemistry / Q000276:immunology*; D019000:Phosphotyrosine / Q000276:immunology; D011485:Protein Binding; D016271:Proto-Oncogene Proteins c-myc / Q000276:immunology; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation / Q000379:methods*; D013997:Time Factors
D000906:Antibodies; D045163:Antibodies, Phospho-Specific; D000939:Epitopes; D010455:Peptides; D016271:Proto-Oncogene Proteins c-myc; D001360:Avidin; D019000:Phosphotyrosine; D001710:Biotin