Optimizing sequence coverage for a moderate mass protein in nano-electrospray ionization quadrupole time-of-flight mass spectrometry.
Sample pretreatment was optimized to obtain high sequence coverage for human serum albumin (HSA, 66.5 kDa) when using nano-electrospray ionization quadrupole time-of-flight mass spectrometry (nESI-Q-TOF-MS). Use of the final method with trypsin, Lys-C, and Glu-C digests gave a combined coverage of 98.8%. The addition of peptide fractionation resulted in 99.7% coverage. These results were comparable to those obtained previously with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The sample pretreatment/nESI-Q-TOF-MS method was also used with collision-induced dissociation to analyze HSA digests and to identify peptides that could be employed as internal mass calibrants in future studies of modifications to HSA.
Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE 68588, USA.
Ryan
R
Matsuda
Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE 68588, USA.
Venkata
V
Kolli
Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE 68588, USA.
Megan
M
Woods
Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE 68588, USA.
Eric D
ED
Dodds
Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE 68588, USA. Electronic address: dhage1@unl.edu.
David S
DS
Hage
D000595:Amino Acid Sequence; D006801:Humans; D010455:Peptides / Q000737:chemistry*; D012709:Serum Albumin / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D010455:Peptides; D012709:Serum Albumin
Collision-induced dissociation; Human serum albumin; Internal calibration; Nano-electrospray ionization quadrupole time-of-flight mass spectrometry; Sample pretreatment; Sequence coverage