IMLS™- A Method for Sequencing Intact Proteins by In-Source
Fragmentation Using Novel "Mass Defect" Labels
M.P. Hall, S. Ashrafi, R. Petesch, and L.V. Schneider
Abstract
We present a new class of "mass defect" tags with broad utility in biomolecular mass
spectrometry. Elements with atomic numbers between 17 (Cl) and 77 (Ir) have a substantially
different nuclear binding energy (mass defect) from the elements common to biomolecules. This
mass defect yields a readily resolvable mass difference between tagged and untagged species in
high-resolution mass spectrometers. Examples of the use of these tags in inverted mass ladder
sequencing (IMLS™) and as isotope differentiated binding energy shift tags (IDBEST™) for
differential peptide display on protein chips are presented. IMLS™ has substantial speed and
cost advantages over traditional tandem MS sequencing methods. IDBEST™ may yield the
precision advantages of isotope-coded affinity tags (ICAT), without the need for affinity cleanup
of the peptides. Mass defect tags also have potential for sequencing other biopolymers (e.g.,
DNA, oligosaccharides, and lipids) and in combinatorial chemistry and high throughput
screening applications.
Introduction
Applications of biomolecular mass spectrometry can be drawn from the areas of
identification and sequencing of proteins,(1) polynucleic acids,(2) and polysaccharides.(3) Mass
spectrometry has been successfully applied to probing biomolecular structure/function
relationships such as protein-ligand and protein-protein interactions.(4) The ability to resolve
stable isotopes has also made mass spectrometry useful for differential display applications.(5)
However, chemical noise in mass spectra arising from matrix impurities, fragmentation products,
or unidentified constituents can compromise spectral analysis. Incorporation of one or more
elements having atomic numbers between 17 (Cl) and 77 (Ir), and more effectively between 35
(Br) and 63 (Eu), into the biomolecules of interest produces a discernible mass shift of these
tagged biomolecules in the mass spectrum away from the rest of the chemical noise, improving
the ability to analyze the spectrum.(6)
The Mass Defect
The mass defect is related to the nuclear binding energy released upon formation and
stabilization of the nucleus of a given isotope.(7) By convention, the mass defect of C-12 is
defined as zero atomic mass units (amu), and the mass defect of any other stable elemental
isotope is calculated as: (8)
Mass Defect (amu) = Monoisotopic Mass - Σ(# Protons and Neutrons)
The mass defects of other elements commonly found in biomolecules (H, N, and O)
differ negligibly from that of carbon and the lower abundance heteroatoms, S and P, exhibit only
slightly larger mass defects. An analysis of the mass defects for the most abundant stable nuclei
of all of the elements shows a maximal mass defect value of approximately -0.1 amu for
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elements with atomic numbers between 35 (Br) and 63 (Eu). Br and I are the easiest of these to
incorporate into organic tags and Br occurs in a natural 50:50 isotope abundance.
Inverted Mass Ladder Sequencing (IMLS™)
IMLS™ is a method for determining an N- or C-terminal protein sequence tag (PST) of
an intact protein by in-source fragmentation in a simple ESI-TOF mass spectrometer (ABI
Mariner™) in less than 2 min.(10) By incorporating a mass defect element into the tag attached
to the N-terminus of myoglobin, the resulting mass defect peptide fragment ladder can be
algorithmically resolved (blue) from the raw fragmentation spectrum (black). The mass defect
tag (shown in inset) incorporates Br, which allows for peak pairing for further resolution of the
tagged b-ion ladder (red) from the residual chemical noise. The first 6 residues are resolvable
with a sequencing algorithm.(11)
Differential Display
Because the spectral deconvolution algorithm preserves the relative abundances of the
two Br isotopes, it is also possible to use isotopically-pure versions of the mass defect tags for
high precision differential display applications. Myoglobin was labeled with isotopedifferentiated binding energy shift tags (IDBEST™), mixed with a whole cell lysate of E. coli.
The mixture was subjected to trypic digestion, desalted with by ZipTip, and spotted on a
Ciphergen SELDI chip. A mass spectrum of this sample was obtained using an MDS Sciex QStar tandem MS equipped with a Ciphergen ionization head. The IDBEST™ tagged myoglobin
peptides were algorithmically recovered (red) from the raw data (white). The sequence of the
tagged N-terminal tryptic peptide was confirmed from the CID fragmentation spectrum.
Conclusions
Mass defect tags are useful for a wide range of biomolecular mass spectrometry
applications because they differentiate the tagged species from other biological molecules. The
examples shown incorporate just a single mass defect element into the label, but up to 4 mass
defect elements incorporated into a tag can be distinguished algorithmically in the mass
spectrum. This allows other biomolecular applications such as sequencing all four bases of DNA
simultaneously and applications in combinatorial chemistry and high throughput screening. The
spectific inverted mass ladder sequencing (IMLS™) application of mass defect tags provides a
100-fold speed and cost advantage over tandem MS sequencing and can be conducted in a
simple ESI-TOF. Isotope-differentiated binding energy shift tags (IDBEST™) can be used for
peptide differential display applications, potentially providing ICAT-like precision without the
need for an affinity clean up step.
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