Top-down protein sequencing and MS3 on a hybrid linear quadrupole ion trap-orbitrap mass spectrometer.
Top-down proteomics, the analysis of intact proteins (instead of first digesting them to peptides), has the potential to become a powerful tool for mass spectrometric protein characterization. Requirements for extremely high mass resolution, accuracy, and ability to efficiently fragment large ions have often limited top-down analyses to custom built FT-ICR mass analyzers. Here we explore the hybrid linear ion trap (LTQ)-Orbitrap, a novel, high performance, and compact mass spectrometric analyzer, for top-down proteomics. Protein standards from 10 to 25 kDa were electrosprayed into the instrument using a nanoelectrospray chip. Resolving power of 60,000 was ample for isotope resolution of all protein charge states. We achieved absolute mass accuracies for intact proteins between 0.92 and 2.8 ppm using the "lock mass" mode of operation. Fifty femtomole of cytochrome c applied to the chip resulted in spectra with excellent signal-to-noise ratio and only low attomole sample consumption. Different protein charge states were dissociated in the LTQ, and the sensitivity of the orbitrap allowed routine, high resolution, and high mass accuracy fragment detection. This resulted in unambiguous charge state determination of fragment ions and identification of unmodified and modified proteins by database searching. Protein fragments were further isolated and fragmented in the LTQ followed by analysis of MS(3) fragments in the orbitrap, localizing modifications to part of the sequence and helping to identify the protein with these small peptide-like fragments. Given the ready availability and ease of operation of the LTQ-Orbitrap, it may have significant impact on top-down proteomics.
Macek
Boris
B
Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany.
Waanders
Leonie F
LF
Olsen
Jesper V
JV
Mann
Matthias
M
D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000737:chemistry; D002417:Cattle; D006801:Humans; D007782:Lactoglobulins / Q000737:chemistry; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational / Q000235:genetics; D040901:Proteomics; D012680:Sensitivity and Specificity; D020539:Sequence Analysis, Protein / Q000295:instrumentation* / Q000379:methods*; D038201:alpha-Crystallins / Q000737:chemistry
D002364:Caseins; D007782:Lactoglobulins; D038201:alpha-Crystallins