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1.  The Development of an Integrated Platform to Identify Breast Cancer Glycoproteome Changes in Human Serum 
Journal of chromatography. A  2009;1217(19):3307-3315.
Protein glycosylation represents one of the major post translational modifications and can have significant effects on protein function. Moreover, changes in the carbohydrate structure are increasingly being recognized as an important modification associated with cancer etiology. In this report, we describe the development of a proteomics approach to identify breast cancer related changes in either concentration and/or the carbohydrate structures of glycoprotein(s) present in blood samples. Diseased and healthy serum samples were processed by an optimized sample preparation protocol using multiple lectin affinity chromatography (M-LAC) that partitions serum proteins based on glycan characteristics. Subsequently, three separate procedures, 1D SDS-PAGE, isoelectric focusing and an antibody microarray, were applied to identify potential candidate markers for future study. The combination of these three platforms is illustrated in this report with the analysis of control and cancer glycoproteomic fractions. Firstly, a molecular weight based separation of glycoproteins by 1D SDS-PAGE was performed, followed by protein, glycoprotein staining, lectin blotting and LC-MS analysis. To refine or confirm the list of interesting glycoproteins, isoelectric focusing (targeting sialic acid changes) and an antibody microarray (used to detect neutral glycan shifts) were selected as the orthogonal methods. As a result, several glycoproteins including alpha-1B-glycoprotein, complement C3, alpha-1-antitrypsin and transferrin were identified as potential candidates for further study.
doi:10.1016/j.chroma.2009.09.029
PMCID: PMC4142217  PMID: 19782370
High performance multi-lectin affinity chromatography; lectin blotting; isoelectric focusing; lectin – antibody microarray
2.  A Proteomics Platform Combining Depletion, Multi-lectin Affinity Chromatography (M-LAC) and Isoelectric Focusing to Study the Breast Cancer Proteome 
Analytical chemistry  2011;83(12):4845-4854.
The discovery of breast cancer associated plasma/serum biomarkers is important for early diagnosis, disease mechanism elucidation and determination of treatment strategy for the disease. In this study of serum samples, a multidimensional fractionation platform combined with mass spectrometric analysis were used to achieve the identification of medium to lower abundance proteins, as well as simultaneously detecting glycan and abundance changes. Immuno-affinity depletion and multi-lectin chromatography (M-LAC) were integrated into an automated HPLC platform to remove high abundance protein and fractionate glycoproteins. The collected glycoproteomes were then subjected to isoelectric focusing (IEF) separation by a digital ProteomeChip (dPC), followed by in-gel digestion and LC-MS analysis using an Orbitrap mass spectrometer. As a result, the total number of identified proteins increased significantly when the IEF fractionation step was included as part of the platform. Relevant proteins with biological and disease significance were observed and the dynamic range of the serum proteome measurement was extended. In addition, potential glycan changes were indicated by comparing proteins in control and cancer samples in terms of their affinity to the multi-lectin column (M-LAC) and the pI profiles in IEF separation. In conclusion, a proteomics platform including high abundance protein depletion, lectin affinity fractionation, IEF separation and LC-MS analysis has been applied to discover breast cancer associated proteins. The following candidates, thrombospondin-1 and 5, alpha-1B-glycoprotein, serum amyloid P-component and tenascin-X, were selected as promising examples of the use of this platform. They show potential abundance and glycan changes and will be further investigated in future studies.
doi:10.1021/ac2002802
PMCID: PMC3148019  PMID: 21513341
3.  A targeted proteomics–based pipeline for verification of biomarkers in plasma 
Nature biotechnology  2011;29(7):625-634.
High-throughput technologies can now identify hundreds of candidate protein biomarkers for any disease with relative ease. However, because there are no assays for the majority of proteins and de novo immunoassay development is prohibitively expensive, few candidate biomarkers are tested in clinical studies. We tested whether the analytical performance of a biomarker identification pipeline based on targeted mass spectrometry would be sufficient for data-dependent prioritization of candidate biomarkers, de novo development of assays and multiplexed biomarker verification. We used a data-dependent triage process to prioritize a subset of putative plasma biomarkers from >1,000 candidates previously identified using a mouse model of breast cancer. Eighty-eight novel quantitative assays based on selected reaction monitoring mass spectrometry were developed, multiplexed and evaluated in 80 plasma samples. Thirty-six proteins were verified as being elevated in the plasma of tumor-bearing animals. The analytical performance of this pipeline suggests that it should support the use of an analogous approach with human samples.
doi:10.1038/nbt.1900
PMCID: PMC3232032  PMID: 21685906
4.  Electron Transfer Ion/Ion Reactions in a Three-Dimensional Quadrupole Ion Trap: Reactions of Doubly and Triply Protonated Peptides with SO2·− 
Analytical chemistry  2005;77(6):1831-1839.
Ion–ion reactions between a variety of peptide cations (doubly and triply charged) and SO2 anions have been studied in a 3-D quadrupole ion trap, resulting in proton and electron transfer. Electron transfer dissociation (ETD) gives many c- and z-type fragments, resulting in extensive sequence coverage in the case of triply protonated peptides with SO2·−. For triply charged neurotensin, in which a direct comparison can be made between 3-D and linear ion trap results, abundances of ETD fragments relative to one another appear to be similar. Reactions of doubly protonated peptides with SO2·− give much less structural information from ETD than triply protonated peptides. Collision-induced dissociation (CID) of singly charged ions formed in reactions with SO2·− shows a combination of proton and electron transfer products. CID of the singly charged species gives more structural information than ETD of the doubly protonated peptide, but not as much information as ETD of the triply protonated peptide.
doi:10.1021/ac0483872
PMCID: PMC1564063  PMID: 15762593
5.  Electron Transfer versus Proton Transfer in Gas-Phase Ion/Ion Reactions of Polyprotonated Peptides 
Journal of the American Chemical Society  2005;127(36):12627-12639.
The ion/ion reactions of several dozen reagent anions with triply protonated cations of the model peptide KGAILKGAILR have been examined to evaluate predictions of a Landau–Zener-based model for the likelihood for electron transfer. Evidence for electron transfer was provided by the appearance of fragment ions unique to electron transfer or electron capture dissociation. Proton transfer and electron transfer are competitive processes for any combination of anionic and cationic reactants. For reagent anions in reactions with protonated peptides, proton transfer is usually significantly more exothermic than electron transfer. If charge transfer occurs at relatively long distances, electron transfer should, therefore, be favored on kinetic grounds because the reactant and product channels cross at greater distances, provided conditions are favorable for electron transfer at the crossing point. The results are consistent with a model based on Landau–Zener theory that indicates both thermodynamic and geometric criteria apply for electron transfer involving polyatomic anions. Both the model and the data suggest that electron affinities associated with the anionic reagents greater than about 60–70 kcal/mol minimize the likelihood that electron transfer will be observed. Provided the electron affinity is not too high, the Franck–Condon factors associated with the anion and its corresponding neutral must not be too low. When one or the other of these criteria is not met, proton transfer tends to occur essentially exclusively. Experiments involving ion/ion attachment products also suggest that a significant barrier exists to the isomerization between chemical complexes that, if formed, lead to either proton transfer or electron transfer.
doi:10.1021/ja0526057
PMCID: PMC1570753  PMID: 16144411
6.  SO2−· Electron Transfer Ion/Ion Reactions with Disulfide Linked Polypeptide Ions 
Multiply-charged peptide cations comprised of two polypeptide chains (designated A and B) bound via a disulfide linkage have been reacted with SO2−· in an electrodynamic ion trap mass spectrometer. These reactions proceed through both proton transfer (without dissociation) and electron transfer (with and without dissociation). Electron transfer reactions are shown to give rise to cleavage along the peptide backbone, loss of neutral molecules, and cleavage of the cystine bond. Disulfide bond cleavage is the preferred dissociation channel and both Chain A (or B)—S· and Chain A (or B)—SH fragment ions are observed, similar to those observed with electron capture dissociation (ECD) of disulfide-bound peptides. Electron transfer without dissociation produces [M + 2H]+· ions, which appear to be less kinetically stable than the proton transfer [M + H]+ product. When subjected to collision-induced dissociation (CID), the [M + 2H]+· ions fragment to give products that were also observed as dissociation products during the electron transfer reaction. However, not all dissociation channels noted in the electron transfer reaction were observed in the CID of the [M + 2H]+· ions. The charge state of the peptide has a significant effect on both the extent of electron transfer dissociation observed and the variety of dissociation products, with higher charge states giving more of each.
doi:10.1016/j.jasms.2005.02.010
PMCID: PMC1356657  PMID: 15914021
7.  Complementary Structural Information from a Tryptic N-Linked Glycopeptide via Electron Transfer Ion/Ion Reactions and Collision-Induced Dissociation 
Journal of proteome research  2005;4(2):628-632.
Glycosylation is an important post-translational modification. Analysis of glycopeptides is difficult using collision-induced dissociation, as it typically yields only information about the glycan structure, without any peptide sequence information. We demonstrate here how a 3D-quadrupole ion trap, using the complementary techniques of collision induced dissociation (CID) and electron-transfer dissociation (ETD), can be used to elucidate the glycan structure and peptide sequence of the N-glycosylated peptide from a fractionated tryptic digest of the lectin from the coral tree, Erythina cristagalli. CID experiments on the multiply protonated glycopeptide ions yield, almost exclusively, cleavage at glycosidic bonds, with little peptide backbone fragmentation. ETD reactions of the triply charged glycopeptide cations with either sulfur dioxide or nitrobenzene anions yield cleavage of the peptide backbone with no loss of the glycan structure. These results show that a 3D-quadrupole ion trap can be used to provide glycopeptide amino acid sequence information as well as information about the glycan structure.
doi:10.1021/pr049770q
PMCID: PMC1350609  PMID: 15822944
glycopeptide; tandem mass spectrometry; ion/ion reaction; electron-transfer dissociation

Results 1-7 (7)