MonoCap HighResolution 2000 Series Optimized for Identification of - PowerPoint PPT Presentation

MonoCap HighResolution 2000 Series Optimized for Identification of Peptides /Proteins for Proteome Research

What is MonoCap HighResolution 2000 ? MonoCap HighResolution 2000 is a 2 meter length monolithic silica capillary column which is designed for identifying extremely high number of peptides/proteins for proteome research via LC-MS/MS.

Physical Properties of MonoCap HighResolution 2000 MonoCap C18 HighResolution 2000 Silica : High Purity Monolithic Silica Gel Through-pore Size : 2 μ m Mesopore Size : 15 nm Bonded Phase : Octadecyl Groups Bimodal Pore Structure of End-capping : Yes MonoCap Internal Diameter : 0.1 mm I.D. Length : 2000 mm Recommended Flow Rate Ranges : 0.3 μ L/min ～ 1 μ L/min Maximum Operating Pressure : 35 MPa, 300 Bar MonoCap HILIC-UP HighResolution 2000 Silica : High Purity Monolithic Silica Gel Through-pore Size : 2 μ m Mesopore Size : 12 nm Bonded Phase : Ureidopropyl Groups End-capping : None Internal Diameter : 0.1 mm I.D. Mesopores Through-pores Length : 2000 mm Recommended Flow Rate Ranges : 0.3 μ L/min ～ 1 μ L/min Maximum Operating Pressure : 35 MPa, 300 Bar Remarks : For identification of highly hydrohophilic peptides/proteins

Why Use MonoCap HighResolution 2000 columns? MonoCap HighResolution Traditional Particle Packed Facts 150 mm columns 2000 mm columns Higher Separation Efficiency Lower More Number of Identified Peptides Less Shorter Total Operation Time Longer Lower Operating Pressure Higher Longer Column Lifetime Shorter Less Total Cost including all the More above factors

Performance of MonoCap C18 HighResolution 2000 MS ： LTQ-Orbitrap XL (Mascot Search) Sample: THP-1 Cell Lysate Tryptic Digest, 5 μ g Number of Column Name Identified Proteins Analysis Time in average MonoCap C18 HighResolution 2000 2,087 10 Hours 0.1 mm I.D. x 2000 mm (2013, 2116, 2131) Particle packed column 680 2 Hours 0.1 mm I.D. x 150 mm (685, 679, 675) All data were acquired, provided by our customer

Performance of MonoCap C18 HighResolution 2000 [1] MonoCap C18 HighResolution 2000 (2000 mm x 0.1 mm I.D.) 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 Time (min) [2] Particle Packed column (3 μ m , 150 mm x 0.075 mm I.D.) Time (min) ● ● ● are analytes having the same molecular weight Conditions System : GLS Capillary HPLC system : [1] 0.5 μ L/min Flow Rate Column : [1] MonoCap C18 High Resolution 2000 (2000 mm x 0.1 mm I.D) [2] 0.3 μ L/min : [2] Particle packed column (3 μ m, 150 mm x 0.075 mm I.D.) : 5 μ L Injection Vol. Trap column : MonoCap C18 Trap Column (50 mm x 0.075 mm I.D.) Detection : MS (TIC m / z 500 - 1500) Eluent : A) 0.1 %HCOOH in CH 3 CN Sample : Tryptic digest of proteins B) 0.1 %HCOOH in H 2 O [1] A / B = 10 / 90 - 600 min - 45 / 55 [2] A / B = 10 / 90 - 180 min - 45 / 55

Performance of MonoCap C18 HighResolution 2000 [1] MonoCap C18 HighResolution 2000 (2000 mm x 0.1 mm I.D.) Time (min) [2] Particle Packed column (3 μ m , 150 mm x 0.075 mm I.D.) Time (min) ● ● ● are analytes having the same molecular weight Conditions System : GLS Capillary HPLC system Flow Rate : [1] 0.5 μ L/min Column : [1] MonoCap C18 High Resolution 2000 (2000 mm x 0.1 mm I.D) [2] 0.3 μ L/min : [2] Particle packed column (3 μ m, 150 mm x 0.075 mm I.D.) Trap column : MonoCap C18 Trap Column (50 mm × 0.075 mm I.D.) Injection Vol. : 5 μ L Eluent : A) 0.1 %HCOOH in CH 3 CN Detection : MS (TIC m / z 500 - 1500) Sample : Tryptic digest of proteins B) 0.1 %HCOOH in H 2 O [1] A / B = 10 / 90 - 600 min - 45 / 55 [2] A / B = 10 / 90 - 180 min - 45 / 55

Performance of MonoCap C18 HighResolution 2000 Analysis Time: 3 hrs Time (min) Analysis Time: 6 hrs 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 Time (min) Analysis Time: 10 hrs 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 Time (min) ● ● ● are analytes having the same molecular weight Column : MonoCap C18 High Resolution 2000 (2000 mm x 0.1 mm I.D) Trap column : MonoCap C18 Trap Column (50 mm x 0.075 mm I.D.) Flow Rate : 0.5 μ L/min Eluent : A) CH 3 CN (0.1%HCOOH), B) H 2 O (0.1%HCOOH) Sample : Tryptic digest of proteins A / B = 10 / 90 - 180, 300, 600 min- 45/55 Injection vol. : 5 μ L

Performance of MonoCap C18 HighResolution 2000 Shotgun Proteomics study using MonoCap C18 HighResolution 2000 mm Sample: Proteins derived from rat serum, brain microsome and T Cell 5 μ L Loop Injection [LTQ-XL] m/z 400-1500 top5 CID [LTQ-Orbtirap XL] m/z 400-1500 top3 CID [mascot search] DB: Refseq_rat, 16776 sequence (including only NP_******) LTQ-XL: cut off =35 pts LTQ-OrbitrapXL: cut off=0.05 All data were acquired, provided by Astellas Pharma Inc., Mr. Hiramoto

Performance of MonoCap C18 HighResolution 2000 Particle Packed 15 cm vs MonoCap C18 HighResolution 2000 Comparison of Number of Identified Proteins MonoCap C18 MonoCap C18 15 cm Offgel fractionator 12hr gradient 6hr gradient Particle packed (12 frs) LTQ-XL LTQ-XL 2hr gradient 15cm packed 2hr (Top5 CID) (Top5 CID) LTQ-OrbitrapXL OrbitrapXL (Top3 CID) (Top3 CID) Rat brain microsome Tryptic digest 1,772 1,661 915 2,800 ca.25 μ g Rat brain microsome Tryptic digest 210 1,487 1,182 - iTRAQ (Top3 HCDCID) 5 μ g Rat serum 0.4 μ L (ca. 20 μ g protein) 329 232 153 - Tryptic digest

Performance of MonoCap C18 HighResolution 2000 Rat brain microsome 5 μ g tryptic digest iTRAQ RT: 0.00 - 119.98 60.80 NL: 68.17 1.75E8 621.6653 588.3217 100 Base Peak 0.1 x 150 mm 3 μ m particle packed column 98.59 MS 416.3369 80 elative Abundance FMBM06_1 72.60 210 protein hit H19_01 644.3710 76.91 99.68 54.15 96.48 60 444.3680 923.5068 (OrbitrapXL, HCDCID top3) 535.3097 581.3677 40 91.58 45.97 77.35 100.85 816.4934 612.2755 599.3589 41.66 415.2116 R 20 560.7780 12.76 31.53 84.38 105.10 0.18 18.59 437.8893 451.7808 951.8036 639.6283 445.1200 445.1200 0 0 10 20 30 40 50 60 70 80 90 100 110 Time (min) RT: 0.00 - 360.01 1 52.34 NL: 0.1 x 2000 mm MonoCap C18 1 .26E7 567.35 1 00 1 65.79 Base Peak 766.50 1 78.50 M S 90 40 ℃ 6hr gradient 924.20 monollith2 80 m_1 1 0901 _ 1 89.57 279.04 04 70 1,187 protein hit 956.83 822.64 ce 1 28.20 1 50.51 n a 60 622.1 3 588.63 d n u 1 38.27 b (LTQ-XL CID top3 ） A 50 652.57 tive 21 2.24 1 24.67 40 285.89 81 7.04 la 579.51 Re 563.40 1 94.99 30 1 03.86 952.1 2 1 1 6.1 8 535.55 224.1 5 20 709.54 259.91 83.75 581 .54 74.82 61 .55 41 6.51 1 0 6.1 5 40.1 6 50.53 61 2.54 235.22 250.42 287.57 31 9.68 335.36 354.51 561 .05 452.09 471 .05 449.96 656.82 444.96 455.47 51 5.97 445.02 445.02 444.94 0 0 20 40 60 80 1 00 1 20 1 40 1 60 1 80 200 220 240 260 280 300 320 340 360 Time (min) RT: 0.00 - 720.02 0.1 x 2000 mm MonoCap C18 NL: 387.38 367.42 7.59E6 766.51 705.56 1 00 40 ℃ 12 hr gradient Base Peak M S 90 451 .80 monollith2 420.82 852.40 80 350.08 1,482 protein hit 675.53 m_1 1 0902_ 567.38 06 70 ce 271 .54 n a 60 (LTQ-XL CID top3 ） d 622.08 n u b 1 91 .38 A 50 255.57 638.1 0 tive 535.58 579.37 501 .49 330.59 403.1 6 40 la 237.52 81 7.06 e 588.57 1 85.1 1 288.92 463.58 R 709.52 30 652.57 952.06 598.1 8 31 4.57 505.79 20 485.90 61 7.25 1 37.24 1 71 .1 0 1 30.21 607.68 746.26 29.49 76.46 220.07 597.39 61 2.59 71 3.60 430.34 470.58 51 6.25 1 0 524.1 4 690.20 704.42 553.71 41 6.55 438.42 452.1 4 848.27 445.1 3 445.1 1 0 0 50 1 00 1 50 200 250 300 350 400 450 500 550 600 650 700 Time (min)

Performance of MonoCap C18 HighResolution 2000 Example of 2-Dimensional LC for Phosphoproteomics Rat T Cell (1x10e8) Tryptic digest from protein GL Sciences’ Purificatin of phosphopeptides via HAMMOC Method Phos-TiO Kit (Phosphoric acid, Ammonia, Pyrrolidine gradual elution) 1/2 1/2 MonoCap 2m 0.1 x 2000 mm MonoCap C18 NanoLC/MS/MS Anti P-Tyr Ab (24hr gradient 、 LTQ-OrbitapXL Injecting two times separately) Pass through Elute 2D LC Identified Phosphopeptides: 11,380 0.1 × 150 mm Silica C18 3 μ m SCX column NanoLC/MS/MS Hours of Operation: 48 hrs (2 x 150mm) （ 2hr gradient 、 LTQ-OrbitapXL ） More identified phosphopeptides 96 fr. in less time compared to 2D LC. 0.1 × 150 mm silicaC18 3 μ m NanoLC/MS/MS (2hr gradient 、 LTQ-OrbitapXL) 267 phosphopeptides Identified Phosphopeptides: 8,324 Hours of Operation: 200 hrs

Performance of MonoCap HILIC-UP HighResolution 2000 For Identification of Highly Hydrophilic Peptides /Proteins Reference: Column Size : 2000 mm x 0.1 mm I.D. Hydrophilic Interaction Chromatography Mass spectrometer : Triple TOF 5600 Using a Meter-Scale Monolithic silica capillary Gradient : Two Gradient Programs, 4 hrs & 8 hrs Column for Proteomics LC-MS, K Horie et al. Anal. Chem . 2014, 86, 3817-3824 4,000 6,500 5,500 HILIC-UP C18