Top-down protein fragmentation by infrared multiphoton dissociation in a dual pressure linear ion trap.
Infrared multiphoton dissociation (IRMPD) was implemented in a novel dual pressure linear ion trap for rapid top-down proteomics. The high pressure cell provided improved trapping and isolation efficiencies while the isotopic profiles of 10+ charged ions could be resolved by mass analysis in the low pressure cell that enabled effective top down protein identification. Striking differences between IRMPD in the low pressure cell and CID in the high pressure cell were observed for proteins ranging from 8.6 to 29 kDa. Because of secondary dissociation, IRMPD yielded product ions in significantly lower charge states as compared to CID, thus facilitating more accurate mass identification and streamlining product ion assignment. This outcome was especially useful for database searching of larger proteins (approximately 29 kDa) as IRMPD substantially improved protein identification and scoring confidence. Also, IRMPD showed an increased selectivity toward backbone cleavages N-terminal to proline and C-terminal to acidic residues (especially for the lowest charge states), which could be useful for a priori spectral predictions and enhanced database searching for protein identification.
Department of Chemistry and Biochemistry, The University of Texas at Austin, 1 University Station A5300, Austin, Texas 78712, USA.
James A
JA
Madsen
Myles W
MW
Gardner
Suncerae I
SI
Smith
Aaron R
AR
Ledvina
Joshua J
JJ
Coon
Jae C
JC
Schwartz
George C
GC
Stafford
Jennifer S
JS
Brodbelt
D030562:Databases, Protein; D007259:Infrared Rays; D017136:Ion Transport; D007477:Ions / Q000737:chemistry*; D011312:Pressure; D011392:Proline / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D007477:Ions; D011506:Proteins; D011392:Proline