New Systems Based on the VersaGrad

Current Binary Configurations Available

Detectors Available 1200 UV/Vis 1 channel programmable 1200 MWD 8 channel programmable 1200 Diode Array Full wavelength spectral capability 1200 Refractive Index 1200 ELSD 1200 Fluorescence

Automation and PC Reactors 1200 Autosampler Alias Autosampler 1200 instant pilot Post Column Reaction system Fract R1 and R2 Fraction collectors Column Oven

Versatility to Meet Any Application Custom build a system by line items: 1. Choose a pump 2. Add a detector (s) 3. Add an injector or Autosampler 4. Add the degasser if desired 5. Add a system accessory kit 6. Add the solvent tray 7. Add EZChrom with control options required 8. Add Instant pilot, PCR or Fraction collector if desired 9. Add a column oven 10. Quote as a package price not by line item

Pre Configured Systems Standard HPLC Systems  Series III Manual with EZChrom  Series 1500 with Autosampler and EZChrom  QGrad with Autosampler PDA and EZChrom Ultra High Pressure Systems  Series III UHP with Autosampler and EZChrom  Series UHP with Autosampler and EZChrom Application Based Systems  Advanced Protein Liquid Chromatography System APLC  Proteome UFFLC System  PCR system for Carbamates

Features  Single and dual piston models available  Pressures up to 6,000psi SS 5,000psi PEEK  Manual or automated injection  Match the performance to the application flows from 0.001ml/min to 100ml/min  Full range of detectors  EZChrom control and data Reduction  Elegance at an affordable price Binary & Quaternary HPLC Systems

VersaGrad Series III Manual Entry level system with EZChrom control & data includes: 1. VersaGrad Series III 2. Degasser & solvent tray 3. EZChrom on preloaded computer 4. Manual injector 5. Standard UV/Vis detector Shown with optional Instant pilot

VersaGrad Series III System with Automation Option Shown with optional autosampler Add other options at any time to upgrade a system Modularity allows reconfiguration for many applications

VersaGrad 1500 Binary System High end automated Binary system containing: 1. VersaGrad Model Autosampler UV detector Degasser 5. Solvent tray 6. PC with EZChrom pre loaded

QGrad Quaternary Gradient System Quaternary high end DAD based system with: 1. QGrad Pump Diode Array Detector Autosampler 4. EZChrome Elite Workstation with DAD Pre installed Degasser & Solvent tray

Features  Single and dual piston models available  Pressures up to 12,000psi Single piston 18,000psi dual piston  A utomated injection  Full range of detectors  EZChrom control and data  Ultimate Performance at an affordable price Ultra High Pressure (UHP) Systems

Series III HP Binary with EZChrom 12,000psi entry level UHP system with: 1. VersaGrad Series III UHP, 12,000psi pump 2. Alias Autosampler UV detector Degasser 5. Solvent tray 6. PC with EZChrom pre loaded

UHP Binary System 18,000 psi ultimate UHP system including: 1. VersaGrad UHP dual piston pump 2. Alias Autosampler UV detector Degasser 5. Solvent tray 6. PC with EZChrom pre loaded

Substantial Savings AssayAnalysis Time Flow ml/min Test per Year Cost per test Cost per 1000 Samples Solvent cost per 1000 Savings per 1000 samples Conventional Isocratic $9.03$9,025.63$1,005 UHP Isocratic $1.50$1,504.27$167$7, Conventional Gradient $45.13$45,128.17$5,024 UHP Gradient $5.26$5,264.95$586$39,863.21

SAVINGS in METHOD DEVELOPMENT  6-10 Times faster scouting runs  System equilibration time is reduced  Solvent changes are faster  You can do in a morning what normally takes days The Ultra High Pressure Binary Systems

UHP Reproducibility 4 Benzoate Esters Column: 1.5micron particle size 2.1mm X 10 cm C angstrom Mobile phase: A Pump Water B Bump Acetonitrile 35%A 65%B Flow: 1ml/min Pressure: 12,500psi Detector: UV at 254nm

UHP Reproducibility – Previous Data

Gradient Reproducibility UHP of Flavors – Lemon Oils Column: 1.5micron particle size 2.1mm X 10 cm C angstrom Mobile phase: A Pump Water B Bump Acetonitrile 30%B to 90%B in 3min Flow: 1ml/min Pressure: 9,000-12,500psi Detector: UV at 214nm

Gradient Reproducibility UHP of Flavors – Previous Data

UHP Binary Pump Retention Time Study at HPLC Pressure Methyl Benzoate Ethyl Benzoate ColumnZorbax C18 5um 4.6 X 15 SolventH20 35% CH3CN 65% Flow1.5ml/mi DetectionUV254nm Pressure1268psi

Methyl Benzoate Ethyl Benzoate Mean1.188Mean Standard Error Standard Error Median1.188Median1.352 Mode1.188Mode1.352 Standard Deviation Standard Deviation Sample Variance4.4444E-07Sample Variance1.16E-06 Kurtosis Kurtosis Skewness E-13Skewness Range0.002Range0.003 Minimum1.187Minimum1.35 Maximum1.189Maximum1.353 Sum11.88Sum Count10Count10 Relative Std Dev0.06% 0.08% New UHP Binary Pump Retention Time Study

Features  Tailored to a given application  18,000psi pumps advantages are exploited in the UFFLC  EZChrom control and data Application Specific Systems

UFFLC of the Proteome Comes standard with 1. VersaGrad UHP 18,000psi pump 2. Alias Autosampler UV detector Degasser 5. Solvent tray 6. PC with EZChrom pre loaded 7. Fract R1 Fraction collector

UFFLC 2-Minute Hemoglobin Rapid Screen SAMPLE: Hemoglobin FASC standard COLUMN: PolyCAT A, 100x2.1-mm; 3-µm, 1500-Å FLOW: 1.2 ml/min A415 Pressure: 6938 psi GRADIENT: 0-0.7’: 18-55% B; ’: 55-85% B; ’: % B; ’: 100% B; 1.4’: Initial A) 40 mM Bis-Tris + 2 mM KCN, pH 6.5 B) 40 mM Bis-Tris + 2 mM KCN mM NaCl, pH 6.8 A 1c F A A2A2 S C A3A3

Minutes mAU Mixed-bed ion-exchange HPLC of yeast lysate: Effect of flow rate COLUMN: PolyCAT A + PolyWAX LP, 200x4.6-mm; 5 µm, 1000-Å GRADIENT: 0 – 0.8 M NaCl in 20 mM HEPES, pH 7.0 A ml/min 4 ml/min

Mixed-bed ion-exchange HPLC of yeast lysate: Effect of flow rate COLUMN: PolyCAT A + PolyWAX LP, 200x4.6-mm; 5 µm, 1000-Å A 280 GRADIENT: (1 ml/min) 0-30’: 0-50 %B; 30-40’: %B; (4 ml/min) 0-2’: 0-8 %B; 2-7’: %B A) 20 mM HEPES, pH 7.0; B) 20 mM HEPES M NaCl, pH 7.0 Minutes A ml/min (1051 psi) 4 ml/min (4709 psi) (X-axis expanded 5x)

Mixed-bed ion-exchange HPLC of E. coli lysate: Fast flow COLUMN: PolyCAT A + PolyWAX LP, 200x4.6-mm; 5 µm, 1000-Å A ml/min 4709 psi GRADIENT: 0-2’: 0-8 %B; 2-5.5’: 8-95 %B; 5.5-6’: %B A) 20 mM HEPES, pH 7.0 B) 20 mM HEPES M NaCl, pH 7.0

PROTEOMICS BY 2-DIMENSIONAL LC/ MS/MS ADVANTAGES OVER 2-D ELECTROPHOPHORESIS 1) Greater dynamic range: Most abundant protein detected Least abundant protein detected 2) Works better for hydrophobic and very basic proteins 3) Handling and automation easier - from Link et al., Nat. Biotechnol. 17 (1999) MudPIT: Multidimensional Protein Identification Technology

# of proteins pIvalue pI Distribution of the Predicted Mouse Proteome From: H. Wang et al., J. Proteome Res. 5 (2006) 361 Acidic proteins; Basic proteins; Use anion-exchange Use cation-exchange

>8 Number of Unique Peptides Number of Proteins PROTEINS IDENTIFIED IN LOW-MOL.-WT. MOUSE SERUM Method: SCX (96 fractions)-RPC Ref: B.L. Hood et al., J. Am. Soc. Mass Spectrom. 16 (2005) 1221 NOT ACCEPTABLE One Hit Wonders. ACCEPTABLE Identifying proteins via 2 peptides or more

Fractionating Intact Proteins Increases Detection of Peptides from Proteins of Low Abundance Low-abundance Protein X is 0.1% of total protein Now Protein X is 1.0 % of total protein in Fraction #6. After digestion, its peptides will be 10x higher a percentage of the total in that fraction than would have been true in a digest of the unfractionated mixture. That greatly increases the chances of identifying Protein X through 2-3 of its fragments rather than just one

Top-Down UHP Proteomics

Histone H4 Acetylation & Methylation Variants COLUMN: PolyCAT A (104CT0315; HILIC mode) VOLTAGE 0 Acetyl 1 Acetyl 2 Acetyl H2A METHYLATION TIME (Min) VOLTAGE 0 4-Acetyl 3-Acetyl 2-Acetyl 1-Acetyl 0-Acetyl H2A METHYLATION (courtesy James Pesavento - U. of Ill.) MITOSIS INTERPHASE The most minor variants can be the most critical

UHP UFFLC Higher throughput than any other system Allows use of standard columns and UHP columns Allows normal chromatography and small scale prep as well as UHP &UFFLC Highest pressure and flow limits on the market today. Why limit yourself FPLC, is now obsolete with the speed and resolution of UFFLC Top down proteomics is now much easier

PCR System Comes standard with 1. VersaGrad Series III Fluorescence detector or UV Degasser 4. Solvent tray 5. PC with EZChrom pre loaded 6. Sensivate post column reaction system 7. Manual injector

Sensivate Post-Column Reactor Applications  Carbamates by OPA  Glyphosates by OPA  Aflatoxin by Iodine  Amino Acids by OPA  Amino Acids by Ninhydrin

PCR System Applications ApplicationReactor 1Temp.Reactor 2 CarbamateNaOH100 o COPA GlyphosateNaOCl40 o COPA AflatoxinsIodine40 o C(not used) CarbohydratesNaOH40 o C(not used) LipidsPhenacyl100 o C(not used) Amino Acids - Ninhydrin Ninhydrin140 o C(not used) Amino Acids - OPANaOCl40 o COPA Amino Acids - PrimaryOPA30 o C(not used)

State of the Art Separations AflatoxinGlyphosateCarbamate

Amino Acids

APLC System Going beyond FPLC 1. VersaGrad Series III UV Detector Degasser 4. Solvent tray 5. PC with EZChrom pre loaded 6. Fract R1 Fraction collector

Mixed-bed ion-exchange HPLC of yeast lysate: COLUMN: PolyCAT A + PolyWAX LP, 200x4.6-mm; 5 µm, 1000-Å A 280 GRADIENT: (1 ml/min) 0-30’: 0-50 %B; 30-40’: %B; A) 20 mM HEPES, pH 7.0; B) 20 mM HEPES M NaCl, pH 7.0 Minutes A ml/min (1051 psi)

TIME (Min) A phosphorylation variants Anion-Exchange of Ovalbumin Phosphorylation Variants COLUMN: PolyWAX LP (#104WX0510) SAMPLE: Sigma Grade VI (99%) GRADIENT: 10 mM K-PO4, pH 7.0; mM NaCl in 20’

Cation-Exchange HPLC of BMP-14 (Bone Morphogenetic Protein 14) COLUMN: PolyCAT A, 100x4.6-mm; 3-µm, 1000-Å (item# 104CT0310; ser# A2562C) MOBILE PHASE: A) 20 mM K-PO4, pH 6.0, with 20% ACN; B) Same M NaCl GRADIENT: 20’ linear, 0-100% B, then 5’ at 100% B 1.0 ml/min A220 SAMPLE: 25 µg BMP-14 in 20 µl of (Mobile Phase A:10 mM HCl = 2:1)

BMP-14: Blowup to show deamidation variants

BMP-14: More gradual gradient to improve separation of variants; resembles preceding growth factor’s profile QUESTION: Do all recombinant growth factors contain ~ 8% Met[O] or mismatched -S-S- variants cysteine knot motif)? New shoulder; deamidation variant? Another new shoulder?

ERLIC of Phosphopeptides with Volatile Solvents Column: PolyWAX LP (104WX0503) Flow rate: 1 ml/min. Detection: A270 (blue) or A260 (red) Gradient: 0-5’: 0%B; 5-45’: 0-100% B; 45-50’: 100%B MP A: 20 mM NH 4 -Formate, pH 2.2, with 70% ACN MP B: [TOP] 100 mM NH 4 -Formate, pH 2.2, w. 64% ACN; [BOTTOM] 20 mM NH 4 -Formate, pH 2.2, w. 10% ACN Which to choose:  [salt] or  [ACN]?

Millivolts Minutes Column: PolyHYDROXYETHYL A (item# 204HY0503), HILIC mode. Detection: A 220 Gradient: 40’ linear, 0-70% B. A) 15 mM TEAP, pH 2.7, with 85% ACN B) " " 10 " SPE OF PEPTIDES: Unexpectedly Selective Extraction by Carbon and C18 NuTips = Transferrin tryptic digest (30 µg) = Peptides extracted/released by NT200C18 from 500 µg digest = " " NT200CAR " " = " " (second set of releasing washes) Urea (1 M)

Comparison of ERLIC vs. HILIC of Amino Acids MOBILE PHASE: 10 mM TEAP, pH 4.0, w. 65% ACN; 1 ml/min TIME (Min) ABSORBANCE (215 nm) PolyWAX LP (basic) D H K R E Cya L I M V P A T pGLU + Q N G S K R H E D Cya N PolyHYDROXYETHYL A (neutral) L V P Q+SQ+S E N R K H D Cya A T G TSKgel Amide-80 (acidic) +M+M I pGlu A T P V pGLU

Systems For Every Application From HPLC to UHP in pressure From UFFLC to Prep in flow Versatile selection of detectors Full automation Data to meet your labs requirements Simplicity and functionality at an affordable price LabAlliance has the system your customer needs