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

allowed absolute quantitation. Comparing chromatograms from variousstages of the 70-hour experiment shows excellent reproducibility. Theaverage number of E. Coli proteins identified was 389±17, in which325 replicated in 20 out of the 35 injections. Moreover, the absolutequantitation measurements using the ADH spike for identified proteinswith >3 peptides is 285±7ng and the quantitation measurement for oneof the other protein spikes, Enolase, shows a CV of 7%.220 Identification of Various Pink-RedPigments Formed by Reacting VariousAmino Acids with Onion (allium cepaL.) Thiosulfinates Using HPLC withDiode Array Detector and TandemMass Spectrometry219 Effective Semi-Automated Extractionof Intact Mitochondria from SolidTissues Using Gentle MechanicalHomogenization and Pressure CyclingTechnologyV. S. Gross 1 , G. Carlson 1 , E. Freeman 2 ,A.R. Ivanov 2 , A. Lazarev 11Pressure BioSciences, Inc., South Easton, MA,United States; 2 HSPH Proteomics Resource, HarvardSchool of Public Health, Boston, MA, United StatesImpaired mitochondrial function has been linked to many diseases,such as stroke, heart disease, cancer, Type II diabetes and Parkinson’sdisease. Mitochondria-enriched preparations are needed forproteomic and metabolomic studies that may provide crucial insightsinto tissue-specific mitochondrial function and dysfunction, andanswer fundamental questions of cell energetic and oxidative stress.Mitochondria extractions from whole tissue samples are typicallyperformed using Potter-Elvehjem homogenizers or similar laborintensivemanual disruption methods that require extensive operatorexperience, and often result in damage to fragile organelles and highsample-to-sample variability. Here we describe a semi-automatedmethod that uses a novel gentle mechanical homogenizer (The PCTShredder) and hydrostatic pressure to release intact mitochondriafrom fresh rat tissues with minimal hands-on time. Pressure CyclingTechnology (PCT)-based tissue homogenization is conducted undercontrolled thermodynamic conditions (time, temperature and pressure)leading to more reproducible results. The quality of mitochondriapreparations was characterized by electron microscopy, 2D PAGE andrespiration assays. Our data demonstrate that mitochondria extractedby the PCT sample preparation system (PCT-SPS) are intact, functional,and exhibit a protein pattern comparable to control samples isolatedusing a conventional Potter-Elvehjem homogenizer. The resultingmitochondria-enriched samples were also subjected to trypsindigestion followed by nanoLC-MS/MS analysis on an LTQ-Orbitrap.Proteomic and pathway profiles of mitochondria samples preparedusing the novel extraction technique were compared to those extractedusing a conventional manual method to demonstrate the purity ofmitochondrial preparations extracted using the novel PCT-SPS method.Y. Rezenom 1 , E. Lee 2 , K. Yoo 2 , D. Russell 1 , B. Patil 21Laboratory for Biological Mass Spectrometry,Department of Chemistry, Texas A&M University,College Station, TX, United States; 2 Vegetable& Fruit Improvement Center, Department ofHorticultural Science, Texas A&M University,College Station, TX, United StatesDuring the processing of onion, pink-red colored pigments are oftenformed. The process is believed to be a multistep process includingenzymatic and non enzymatic reactions. In order to investigate thisprocess, we developed a reaction system, where pink-red pigments(‘pinking’) can be formed by reacting amino acids with onionthiosulfinate formed by reacting an isolated garlic alliinase and (+)-S-1-propenyl-L-cysteine sulfoxide (1-PeCSO) in the natural onion juice. Theunknown pink-red pigments formed during this process were separatedand detected using a high-performance liquid chromatography (HPLC)and a diode array detector (DAD) at 515 nm. Fractions collected fromthis separation were further analyzed using liquid chromatography(LC) and tandem mass spectrometry. Similar head group structure, two3,4-dimethyl pyrrolyl rings that were cross linked by allyl group, wasdetermined for all conjugate-pigments formed from different aminoacids based on the accurate and tandem mass spectrometry. However,the tail group attached to the N-terminal of pyrrole ring differed foreach pink-red pigment depending on the amino acid used. In addition,in most cases more than one pink compound were identified for thesame amino acid used. We presumed that the complexity of the pinkredpigments was due to the involvement of 21 natural amino acids andother derivatives of the products. Finally, we suggest that the pinkingprocess in crushed onion is very similar to the greening process incrushed garlic, emphasizing that thiosulfinates from flavor precursorsand free amino acids are absolutely necessary during the discoloration.221 Improvements in Data-DependentAcquisition for Enhanced ProteinIdentificationC. Miller, N. Kitagawa, W. Tang, J. Roark,J. Satulovsky, P. PerkinsAgilent Technologies, Palo Alto, CA, United StatesPoster AbstractsComprehensive proteome analysis can be very challenging due tocomplexity and range of protein concentrations. Techniques such as2D LC, pI-based fractionation and gel electrophoresis are typicallyemployed to increase separation efficiency as a strategy for obtainingmore peptide MS/MS spectra and thus increasing the number of proteinsidentified. In data-dependent mode, efficient and comprehensiveprotein identification depends on several conditions during theacquisition, among them: the number of precursors examined per unittime, selection of the most promising precursors for fragmentation,and application of the appropriate fragmentation conditions to yieldthe highest quality product ion spectrum during acquisition. This workdescribes changes to the precursor selection and fragmentation stepsto select the precursors most likely to produce good quality peptideMS/MS spectra, and to increase the quality of those spectra.ABRF 2011 — Technologies to Enable Personalized Medicine • 93