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 제 목 PCT News Volume 5 , PBI Discovery Starts With Sample Prep 
 Volume 5 , Issue 5 May 2011 
 Pressure Cycling Technology ( PCT ) 
 Cited at Scientific Meetings by Third Party Scientists 
 Composition of Microbial 
 Jamie Scott , Debarati Paul , Magan Green , Lewis R . Brown , 
 Mark L . Lawrence 
 College of Veterinary Medicine , Biological Sciences , 
 Mississippi State University , Mississippi State , MS - 39762 
 Microbial communities in the subterranean environment are unique in that they are completely isolated from biological communities that rely on photosynthesis , Earth  s atmosphere , or oceans . Interestingly , bacteria found in petroleum deposits are in a dormant form known as Ultramicrobacteria because petroleum deposits are very limited in nitrogenous and phorous - containing nutrients . These are unique populations as compared to other subsurface microbial communities and might consist of novel species , new metabolic capabilities , and undiscovered adaptive microbial mechanisms . They are cultured using methods for oligotrophic organisms in the lab . The goal of the current study was to isolate and identify cultivable UMBs based on their hydrocarbon - utilizing properties and also to evaluate microbial community structure in petroleum deposits using 16 S rDNA techniques such as ARDRA ( amplified ribosomal DNA restriction analysis ) of the environmental DNA extracted from the cores . 
 CALENDAR OF PBI EVENTS 
 59 th ASMS 
 and Allied Topics Midwest LIRG Meeting 
 Research : From 
 June 5 - 9 2011 
 Abstracts of Posters Expected 
 the 59 th ASMS Conference on Mass 
 Spectrometry and Allied Topics , to 
 be held June 5 - 9 , 2011 at the 
 Colorado Convention Center in 
 The Evaluation of Pressure - Assisted Enzymatic Digestion for the Optimal Digestion of Monoclonal Antibodies 
 Centocor R & D , a division of J & JPRD LLC , Radnor , PA 
 MP 11 - LC - MS : Sample Preparation - Peptides , poster number : 201 , Monday , Poster Hall 
 Novel Aspect : Reducing the time required for the characterization of therapeutic mAbs by bottom - up approaches . 
 The characterization of monoclonal antibodies ( mAbs ) using peptide mapping is achieved through a combination of top - down and bottom - up approaches . For bottom - up studies , the mAb is typically digested with either trypsin or Lys - C , and the resulting peptides analyzed by HPLC - MS / ( MS ) . In - solution digestion is the most common approach to achieve this goal , as sequence coverage and the assessment of low - level post - translational modifications is often required . Although effective , the in - solution digestion protocol is time consuming and overnight digestion is usually required . Pressure - assisted enzymatic digestion ( PAED ) is an emerging technology used to achieve comparable , or in some cases better , digestion efficiency in significantly less time . In this study , the effectiveness of PAED is evaluated at different conditions using an anti - Respiratory Syncytial Virus ( anti - RSV ) mAb . 
 The PAED was performed using a Pressure Biosciences NEP 2320 Barocycler under two different conditions . In the first condition ( native ) , the samples were mixed with enzyme , while in the second condition , the samples were first reduced and alkylated ( R & A ) on the bench using standard protocols , then mixed with enzyme . Replicate samples were digested in the Barocycler for various intervals ( 2 hr max ) using defined sequences of alternating high and low 
 Composition of Microbial Communities in Petroleum Deposits : Continued from Page 1 
 MetagenomicDNA : 
 The PowerMax Soil DNA Isolation Kit ( MoBio Laboratories , CA ) was adapted for use with the Pressure Cycling Technology ( PCT ) Barocycler Instrument ( Pressure BioSciences , MA ) ( 2 ) . One gram of the crushed core material was vortexed with PowerBead buffer then mixed with lysis buffer and vortexed to mix . The PULSE Tube ( Pressure BioSciences , MA ) was loaded into the Barocycler and run for 80 cycles ( 35 kpsi for 20 sec at pressure , and 5 sec at ambient pressure which lysed bacteria using high pressure . DNeasy spin column ( QIAGEN ) was used to purify the DNA from the cell debris . The DNA was quantified and diluted with water for PCRs . 
 Figure 2 . ARDRA gels showing RsaI digestion pattern of bacterial 16 S rDNA clones 
 The core was populated with an unexpectedly diverse range of bacteria . 
 PCT technology proved invaluable in isolation of metagenomic DNA . 
 BLAST searches indicated much of the core population is similar to the microbial population from the Yellow Sea , followed by similarity to microbial mats of hypersaline lakes and also to some gut microflora . 
 Several phylotypes belonged to ? - proteobacteria , which commonly exist in all environments . 
 Click Here for Complete Poster 
 The Evaluation of Pressure - Assisted Enzymatic Digestion for the Optimal Digestion of Monoclonal Antibodies : Continued from Page 1 
 pressure . Replicate 18 hr in - solution digestion was also performed under both native and R & A conditions ( with or without denaturation ) as controls . All digests were analyzed using a Waters Acquity UPLC coupled with a Waters Premier Qq / TOF in DDA mode . Sequence coverage was determined by MASCOT search against an in - house database . 
 The preliminary results indicate the superiority of the PAED approach for the rapid enzymatic digestion of proteins . Sequence coverage of greater than 90 % was achieved from both the native and the R & A samples within 60 min in the Barocycler . The results obtained using the 60 min PAED approach were comparable to those obtained from the control sample that was denatured , reduced and alkylated followed by overnight digestion . When the model mAb sample was only reduced and alkylated , without the denaturing step , no peptides were identified under the same database search criteria . This indicates the effectiveness of the PAED approach in reducing the total cycle time required for the characterization of mAbs . Moreover , the reproducibility of the PAED approach was confirmed by replicate analysis . In this study , the effect of the different experimental conditions on various success criteria , including but not limited to , sequence coverage , number of missed cleavages and the number of unmatched peptides , was investigated and will be presented . 
 Does LC Separation of Intact Proteins Hinder Identification of Bacterial Markers ? 
 Steven M . Musser 
 FDA - CFSAN , College Park , MD 
 MP 33 - Intact Proteins : Sequence Analysis , poster number : 564 , Monday , Poster Hall 
 Novel Aspect : Comparison of detected intact masses , top - down , and bottom - up for identification of strain specific bacterial markers . 
 Intact protein expression profiling and subsequent identification of marker proteins is a powerful tool for bacterial strain differentiation . Advances in protein chromatography and deconvolution software make it possible to generate reproducible accurate mass  retention time profiles of > 500 features per strain . Advances in top - down technology and informatics make it possible to identify > a hundred proteins per sample by online LC - MS / MS , while maintaining intact mass for identification of SNP based biomarkers . 
 Abstracts of Posters Expected to be Presented at the 59 th ASMS : Continued from Page 2 
 However , protein level LCMS biases against classes and sizes of proteins and bottom - up proteomics identifies ten times the number of proteins . We investigate limits to mapping intact masses to bottom - up protein identifications , SNP identification by top - down versus bottom - up , and which proteins are lost with protein chromatography . 
 Salmonella proteins are extracted with a Barocycler ( Pressure Biosciences ) and separated with a Prosphere P - HR column ( 150 mm x 2 . 1 mm ) . LCMS was performed on a QTOF Premier ( Waters ) and deconvoluted to accurate mass , retention time , and intensity profiles with ProteinTrawler 6 ( BioAnalyte ) . Top - down and bottom - up MS / MS are performed on an Orbitrap ( ThermoFisher ) using a TriVersa Nanomate source ( Advion Biosciences ) . LCMS fractions were infused for top - down identification or digested and analyzed by LC - MS / MS . On - line top - down LC - MS / MS was also performed using the same LC gradient to match retention times . The identical lysate was run on a 1 D SDS - PAGE gel , digested and analyzed by LC - MS / MS . Bottom - up spectra were searched with the Mascot algorithm and top - down spectra were searched with the ProSightPC algorithm . 
 LCMS data show good protein level separation and reproducible extraction of protein molecular weight and retention time profiles , as well as the presence of type specific bacterial marker masses . The one hundred most abundant proteins from intact protein profiles have been identified by on - line and off - line top - down MS / MS . Bottom - up digestion of the corresponding LC fractions yields significantly more identifications , but database deficiencies mean those identifications can not always be mapped back to the intact mass . This can complicate definitive identification of differential protein profile marker masses . However , bottom - up analysis of fractions has the benefit of identifying proteins that may be chromatographically separated but not detected or resolved by intact protein LCMS . Both of these experiments limit bacterial strain analysis to the proteins that are amenable to intact protein chromatography . GeLC analysis from SDS - PAGE digestions yielded many additional identifications from the bottom - up , most of which were in a mass range not amenable to top - down MS / MS , were present at an abundance that is too low for intact protein mass spectrometry , or did not separate on the column . However , while SDS - PAGE maintains an approximate link to the intact protein mass , the comparatively small mass shifts induced by SNP differences between different strains can not be resolved by the gel . Without an intact mass measurement , we are dependent on the database and peptide sequence coverage for detection of the presence of SNPs . 
 Continued on Page 3 Column B 
 Does LC Separation of Intact Proteins Hinder Identification of Bacterial Markers ? ( continued from Page 3 Column A ) 
 Considering the diversity of bacteria , relatively few strain specific bacterial proteins are found in protein sequence databases . On the other hand , the mass range and total number of proteins identified by GeLC analysis surpasses the number of proteins amenable to intact protein HPLC separation . We will also investigate the difficulty in referencing proteins identified by searching bottom - up data against incompletely annotated databases back to an intact mass . 
 Development of a 20 kpsi Enzymatic Digester for High Throughput Proteomic Analysis and Its Application to Membrane Proteomics 
 ThP 11 - Drug Metabolism : High Throughput / Robotics , poster number : 211 , Thursday , Poster Hall 
 Novel Aspect : A new automated high pressure online enzymatic digestion reactor provides comparable digestion to overnight methods in just 20 min . 
 Digesting proteins into peptides is an essential step in bottom - up proteomics . Recently , pressure assisted digestion using pressure cycling technology ( PCT ) was demonstrated to significantly speed time needed for digestions from hours to minutes . In this work , we developed a novel ultra - high pressure PCT reactor that couples to a 2 - column LC - MS platform . The reactor consists of a pressure generator , a temperature controlled flow - through pressure cell , isolated from the flow path by high pressure valves . The system was tested initially with a pool of standard proteins and later with a set of digestion resistant proteins , including the hydrophobic membrane - spanning protein bacteriorhodpsin ( Halobacterium halobium ) and a water - insoluble fraction of Shewanella oneidensis proteins . 
 The reactor was coupled inline between the injection valve and a C 18 trapping column on the LC system . Samples were mixed with trypsin and split into aliquots : part of the sample was subjected to an overnight digestion ( control ) , while the 5 uL aliquot was passed into the PCT reactor and pressurized at 20 , 000 psi for 20 min , after which the digested sample was either captured on a C 18 trapping column and subjected online to LC - MS / MS using an LTQ - FT or collected for subsequent LC - MS / MS analysis . A commercial four protein standard , bacteriorhodopsin , and an insoluble fraction of Schewanella oneidensis were used in this study . Peptide identification rates were used to evaluate the digestion efficiency . 
 Abstracts of Posters Expected to be Presented at the 59 th ASMS 
 Development of a 20 kpsi Enzymatic Digester for High Throughput Proteomic Analysis and Its Application to Membrane Proteomics : Continued from Page 3 Column B 
 Identification and Validation of Vaccine Candidate Proteins by 2 - Dimensional Mass Spectrometry Analysis of Group A Streptococcus 
 Merck and Co . , West Point , PA 
 ThP 35 - Protein Therapeutics : Quantitative Analysis , poster number : 634 , Thursday , Poster Hall 
 Novel Aspect : A comparative proteomic approach for identifying and validating bacterial surface - exposed proteins by mass spectrometry for a vaccine against GAS . 
 Surface proteins of bacteria are involved in adhesion and invasion of host cells . Since surface proteins likely interact 
 Continued on Page 4 Column B 
 Identification and Validation of Vaccine Candidate Proteins by 2 - Dimensional Mass Spectrometry Analysis of Group A Streptococcus : Continued from Page 4 Column A 
 with the host immune system , they are important for vaccine discovery research . We employed on - line multidimensional protein identification technology to characterize the surface of group A Streptococcus ( GAS ) . Surface proteins were selectively digested with trypsin after mutanolysin and lysozyme treatments of intact bacteria . The obtained surface proteome was compared to the whole cell proteome obtained by analyzing total cell lysate . PSortB , a protein prediction algorithm for subcellular localization , was applied to demonstrate enrichment of proteins predicted to be surface localized . We show that identified surface proteins include the most protective antigens reported in literature and 10 newly discovered potential vaccine candidates . 
 For surface digestion , strains M 1 or M 3 GAS cells were brought up in 20 % sucrose in Tris - EDTA pH 8 . 0 . They were treated with mutanolysin and lysozyme , followed by digestion with trypsin for 0 . 5 h at 37 ° C . For total cell lysate digestion , strain M 1 GAS was lysed using pressure cycling technology ( Pressure Biosciences NEP 2320 ) . Extracted proteins were reduced and alkylated , then digested in urea using lysC , followed by trypsin . MS data was acquired on a LTQ ion trap instrument ( Thermo Fisher ) operated in profile mode . 5 µ g of peptides were separated on - line by strong cation exchanger chromatography followed by C 18 reversed phase nano - LC using 10 discrete salt steps . MS / MS scans were performed on the top 7 precursor ions in reverse order . 
 690 proteins were identified in the whole cell lysate of M 1 . 4 . 9 % of total MS signal intensity was comprised of signals from proteins predicted by PSortB as cell wall or non - cytoplasmic proteins . 12 . 7 % of the total MS signal intensity was comprised of signals from proteins of unknown localization . A total of 305 and 334 proteins were identified in the surface digest of strains M 1 and M 3 , respectively . The portion of mass spectrometry signal contributed by surface proteins identified by PSortB was 41 % for the M 1 strain and 27 % for the M 3 strain . The > 8 - fold enrichment of surface proteins for GAS M 1 was reproduced with a > 5 - fold enrichment of surface proteins for GAS M 3 . The M protein , type 1 ( NT 01 SP 1656 ) has been described in the literature to be the highest abundant surface protein in group A Streptococcus . M protein was also identified as the highest abundant protein for both GAS strains M 1 and M 3 in our measurements . The described comparative proteomics method identified ten potential vaccine candidates and allowed us , in addition , to follow the expression state for eleven proteins that had been previously identified by molecular biology . 
 18 PCT News Volume 5 , PBI Discovery Starts With Sample Prep 관리자 2011 . 06 . 07 3904 
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