Conference Program - ABRF 2011 - Association of Biomolecular ...

MuDPIT, and off-gel electrophoresis (OGE) ahead of LC-MS/MS.We have evaluated an alternative method that does not requireadditional sample fractionation on additional equipment, meanwhileminimizing alterations introduced by analytical techniques. For bothwhole human cells and isolated mitochondria, the proposed methodinvolved exposure of the pelleted cells or organelles to sequencegradetrypsin in order to obtain peptides and proteins cleaved fromthe cell and organelle surfaces. The “stripped” cells and mitochondriawere also lysed and their contents digested. Additionally, total lysatesof cells and mitochondria not exposed to trypsin ahead of lysis weregenerated and tryptically digested. The obtained proteomic profilingresults were compared to those acquired using commonly employedfractionation techniques (OGE and SDS-PAGE). Resulting digests fromall protocols were analyzed using LC-MS/MS, and all were analyzedfor the proportion of unique and differentially enriched proteinsachieved with each. Our results showed that the inexpensive andsimplistic fractionation method allowed for similar or superior depthof proteomic profiling as well as a substantial increase in the numberof unique identifications of membrane-bound and extracellular matrixrelated proteins. Additionally, the method provides information aboutspatial localization and differential abundance of proteome subsetsin the cell or organelle architectural compartments. These resultssuggest that a very straightforward approach for the preparation ofcellular and organellar specimens prior to proteomic characterizationcan be reproducibly and reliably utilized by biological and medicallaboratories that have no previous analytical biochemistry expertise.232 Detecting Immune System ResponseProteins in a 500 Year-old IncaMummyA. Koller 6 , A. Corthals 1 , L. Davalos 2 ,D.W. Martin 3,4 , R. Rieger 3 , E.I. Chen 3,51City University of New York, John Jay College ofCriminal Justice, Department of Sciences, New York,NY, United States; 2 Department of Ecology andEvolution and Consortium for Inter-DisciplinaryEnvironmental Research, State University of NewYork Stony Brook, Stony Brook, NY, United States;3Stony Brook Proteomics Center, State Universityof New York Stony Brook, Stony Brook, NY, UnitedStates; 4 Department of Medicine, Division ofHematology, State University of New York StonyBrook, Stony Brook, NY, United States; 5 StateUniversity of New York Stony Brook, Department ofPharmacological Sciences, Stony Brook, NY, UnitedStates; 6 State University of New York Stony Brook,Stony Brook, NY, United StatesDisease detection in ancient human samples currently relies ongenomic-based assays, which are error prone due to contamination andcannot distinguish between active and latent pathogenic infection. Onthe other hand, protein-based assays such as global protein profilingoffer complementary alternatives for the pathological diagnosis ofarcheological specimen. The discovery of three Inca mummies in 1998,perfectly preserved in the permafrost of the high Andes, allowed us toanalyze mummy samples by protein-based and genomic-based assay.A buccal swab from one of the 500 year old mummy was analyzed byshotgun proteomics to detect the protein profile. Among the identifiedproteins, we found a signature of proteins indicating an immuneresponse to a bacterial infection at the time of the mummy’s death.Based on the external visible symptoms and the gamut of immuneresponse proteins obtained from the mouth swab, we suspectedthat the pulmonary infection was caused by Mycobacterium. PCRassay followed by direct sequencing of the PCR products confirmedthe presence of Mycobacterium sp. in the mouth swab. Until now,immunoassays have been the only way to detect an active immuneresponse and infer infection in historical samples, but these wereplagued by low specificity and sensitivity. However, we demonstratehere the feasibility of incorporating global protein profiling in thediagnosis of infection from archeological samples. Protein signaturesobtained from these samples could be extremely useful in determiningthe status of infection while genomic-based assays can be used todetect the identity of the pathogen.233 A New Technology for HighlyEfficient and Reproducible AlbuminDepletion from Whole SerumC. Bolcato, H. Anderson, M. Maust, G. Boyce,M.J. PowellProtea Biosciences, Morgantown, WV, United StatesOne of the major problems faced in the analysis of the human serumproteome is the broad dynamic range of its protein constituents.High abundance proteins, such as human serum albumin (HSA) andgamma-immunoglobulin (IgG), which together comprise 75% of totalserum protein content, inhibit the analysis of lower abundant proteinsof interest. Affinity-based depletion strategies are often employed toselectively remove these high abundance proteins in order to enrichthe lower abundant proteins and facilitate their analysis. Traditionally,antibody-based schemes are used to produce affinity-binding ligandsfor HSA and IgG. Variability in the production of these antibodies leadsto a wide variety of specificities and selectivities. Thus, the efficienciesfor antibody-based depletion strategies for the selective removalof high abundance proteins in human serum can vary broadly, from70 to 95%, and can dramatically affect the reproducibility of humanserum proteome studies. Here, a new approach to affinity-based serumalbumin removal is presented. Instead of utilizing a traditional antibodybaseddepletion approach, the ProteaPrep albumin depletion captureligand is a recombinant bacterial protein that is highly purified forrobust, efficient removal of albumin from human serum samples. Therecombinant ligand technology produces binding constants withhuman serum albumin (Kd ~ 1 × 10-11M) that are significantlystronger than antibody-based methods, which have binding constantsthat range from Kd’s of 1 × 10-6 to 1 ×10-8 M. The resultsshow that the functionalized beads bind and remove albumin fromserum in a highly efficient and rapid manner. Depletion efficiencies are>99% for the selective removal of HSA from serum samples in less than20 minutes. The recombinant bacterial protein ligands are shown to beeffective for the depletion of albumin from the sera of other species,including mouse, rat, pig, horse, cow, and monkey.234 Qualitative and QuantitativeCharacterization of Proteomes UsingIon Mobility Mass Spectrometry withData Independent AcquisitionM. Stapels, K. Fadgen, S. Geromanos, J. LangridgeWaters Corporation, Milford, MA, United StatesA central goal in a proteomics study is to fully characterize a sample bothqualitatively and quantitatively. A data-independent analysis yieldsreproducible fragmentation and peak area information for peptides inPoster AbstractsABRF 2011 — Technologies to Enable Personalized Medicine • 97