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

MonoCap HighResolution 2000 Series

Optimized for Identification of Peptides /Proteins for Proteome Research

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.

What is MonoCap HighResolution 2000 ?

MonoCap C18 HighResolution 2000

Silica : High Purity Monolithic Silica Gel Through-pore Size : 2 μm Mesopore Size : 15 nm Bonded Phase : Octadecyl Groups End-capping : Yes Internal Diameter : 0.1 mm I.D. Length : 2000 mm Recommended Flow Rate Ranges : 0.3 μL/min ～ 1 μL/min Maximum Operating Pressure : 35 MPa, 300 Bar

Physical Properties of MonoCap HighResolution 2000

MonoCap HILIC-UP HighResolution 2000

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

Why Use MonoCap HighResolution 2000 columns?

Facts

Separation Efficiency

Higher

Lower Number of Identified Peptides

More

Less Total Operation Time

Shorter

Longer Operating Pressure

Lower

Higher Column Lifetime

Longer

Shorter Total Cost including all the

Less

More above factors

MonoCap HighResolution 2000 mm columns

Traditional Particle Packed 150 mm columns

Performance of MonoCap C18 HighResolution 2000

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

All data were acquired, provided by our customer

Performance of MonoCap C18 HighResolution 2000

180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 160 140 120 100 80 60 40 20

[1] MonoCap C18 HighResolution 2000 (2000 mm x 0.1 mm I.D.)

Time (min)

[2] Particle Packed column (3 μm , 150 mm x 0.075 mm I.D.)

Time (min)

Conditions System Column Trap column Eluent : GLS Capillary HPLC system : [1] MonoCap C18 High Resolution 2000 (2000 mm x 0.1 mm I.D) : [2] Particle packed column (3 μm, 150 mm x 0.075 mm I.D.) : MonoCap C18 Trap Column (50 mm x 0.075 mm I.D.) : A) 0.1 %HCOOH in CH3CN B) 0.1 %HCOOH in H2O [1] A / B = 10 / 90 - 600 min - 45 / 55 [2] A / B = 10 / 90 - 180 min - 45 / 55 : [1] 0.5 μL/min [2] 0.3 μL/min : 5 μL : MS (TIC m / z 500 - 1500) : Tryptic digest of proteins Flow Rate Injection Vol. Detection Sample

• ● ● are analytes having the same molecular weight

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 μm , 150 mm x 0.075 mm I.D.)

Time (min)

• ● ● are analytes having the same molecular weight

Conditions System Column Trap column Eluent : GLS Capillary HPLC system : [1] MonoCap C18 High Resolution 2000 (2000 mm x 0.1 mm I.D) : [2] Particle packed column (3 μm, 150 mm x 0.075 mm I.D.) : MonoCap C18 Trap Column (50 mm × 0.075 mm I.D.) : A) 0.1 %HCOOH in CH3CN B) 0.1 %HCOOH in H2O [1] A / B = 10 / 90 - 600 min - 45 / 55 [2] A / B = 10 / 90 - 180 min - 45 / 55 : [1] 0.5 μL/min [2] 0.3 μL/min : 5 μL : MS (TIC m / z 500 - 1500) : Tryptic digest of proteins Flow Rate Injection Vol. Detection Sample

Analysis Time: 3 hrs

180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 160 140 120 100 80 60 40 20 180 200 220 240 260 280 300 320 340 360 160 140 120 100 80 60 40 20

Performance of MonoCap C18 HighResolution 2000

Time (min)

Analysis Time: 6 hrs

Time (min)

Analysis Time: 10 hrs

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 μL/min Eluent : A) CH3CN (0.1%HCOOH), B) H2O (0.1%HCOOH) A / B = 10 / 90 - 180, 300, 600 min- 45/55 Sample : Tryptic digest of proteins Injection vol. : 5 μL

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 μL 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

MonoCap C18 12hr gradient LTQ-XL (Top5 CID) MonoCap C18 6hr gradient LTQ-XL (Top5 CID) 15 cm Particle packed 2hr gradient LTQ-OrbitrapXL (Top3 CID) Offgel fractionator (12 frs) 15cm packed 2hr OrbitrapXL (Top3 CID) Rat brain microsome Tryptic digest ca.25 μg 1,772 1,661 915 2,800 Rat brain microsome Tryptic digest iTRAQ 5 μg 1,487 1,182 210 (Top3 HCDCID)

• Rat serum 0.4 μL

(ca. 20 μg protein) Tryptic digest 329 232 153

• Particle Packed 15 cm vs MonoCap C18 HighResolution 2000

Comparison of Number of Identified Proteins

Performance of MonoCap C18 HighResolution 2000

RT: 0.00 - 119.98 10 20 30 40 50 60 70 80 90 100 110 Time (min) 20 40 60 80 100 R elative Abundance 60.80 621.6653 68.17 588.3217 98.59 416.3369 72.60 644.3710 99.68 444.3680 76.91 923.5068 54.15 535.3097 96.48 581.3677 91.58 816.4934 45.97 612.2755 77.35 599.3589 100.85 415.2116 41.66 560.7780 31.53 451.7808 84.38 951.8036 12.76 437.8893 105.10 639.6283 0.18 445.1200 18.59 445.1200 NL: 1.75E8 Base Peak MS FMBM06_1 H19_01

RT: 0.00 - 720.02 50 1 00 1 50 200 250 300 350 400 450 500 550 600 650 700 Time (min) 1 20 30 40 50 60 70 80 90 1 00 R e la tive A b u n d a n ce 387.38 766.51 367.42 705.56 451 .80 852.40 420.82 675.53 350.08 567.38 271 .54 622.08 1 91 .38 535.58 255.57 579.37 638.1 403.1 6 501 .49 81 7.06 330.59 588.57 237.52 709.52 288.92 652.57 463.58 952.06 1 85.1 1 598.1 8 505.79 746.26 31 4.57 607.68 485.90 71 3.60 61 7.25 430.34 1 37.24 61 2.59 1 71 .1 470.58 1 30.21 51 6.25 597.39 41 6.55 220.07 553.71 76.46 452.1 4 29.49 438.42 524.1 4 848.27 704.42 445.1 1 690.20 445.1 3 NL: 7.59E6 Base Peak M S monollith2 m_1 1 0902_ 06 RT: 0.00 - 360.01 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) 1 20 30 40 50 60 70 80 90 1 00 Re la tive A b u n d a n ce 1 52.34 567.35 1 65.79 766.50 1 78.50 924.20 1 89.57 956.83 279.04 822.64 1 28.20 622.1 3 1 50.51 588.63 1 38.27 652.57 21 2.24 81 7.04 1 24.67 579.51 285.89 563.40 1 94.99 952.1 2 1 03.86 535.55 1 1 6.1 8 709.54 224.1 5 581 .54 259.91 41 6.51 83.75 61 2.54 74.82 561 .05 61 .55 452.09 250.42 449.96 50.53 471 .05 287.57 656.82 235.22 51 5.97 40.1 6 455.47 335.36 445.02 6.1 5 444.96 354.51 444.94 31 9.68 445.02 NL: 1 .26E7 Base Peak M S monollith2 m_1 1 0901 _ 04

0.1 x 150 mm 3 μm particle packed column 210 protein hit (OrbitrapXL, HCDCID top3)

Rat brain microsome 5 μg tryptic digest iTRAQ

0.1 x 2000 mm MonoCap C18 40 ℃ 6hr gradient 1,187 protein hit (LTQ-XL CID top3） 0.1 x 2000 mm MonoCap C18 40 ℃ 12 hr gradient 1,482 protein hit (LTQ-XL CID top3）

Performance of MonoCap C18 HighResolution 2000

Example of 2-Dimensional LC for Phosphoproteomics

Rat T Cell (1x10e8) Tryptic digest from protein

0.1 x 2000 mm MonoCap C18 NanoLC/MS/MS (24hr gradient、LTQ-OrbitapXL Injecting two times separately)

Purificatin of phosphopeptides via HAMMOC Method (Phosphoric acid, Ammonia, Pyrrolidine gradual elution) Anti P-Tyr Ab Elute Pass through

0.1 × 150 mm silicaC18 3μm NanoLC/MS/MS (2hr gradient、LTQ-OrbitapXL) SCX column (2 x 150mm) 0.1 × 150 mm Silica C18 3 μm NanoLC/MS/MS （2hr gradient、LTQ-OrbitapXL）

96 fr.

Identified Phosphopeptides: 11,380 Hours of Operation: 48 hrs 267 phosphopeptides

2D LC MonoCap 2m

1/2 1/2

More identified phosphopeptides in less time compared to 2D LC. GL Sciences’ Phos-TiO Kit Identified Phosphopeptides: 8,324 Hours of Operation: 200 hrs

For Identification of Highly Hydrophilic Peptides /Proteins

Performance of MonoCap HILIC-UP HighResolution 2000 6,500 5,500 4,000 HILIC-UP C18

Column Size : 2000 mm x 0.1 mm I.D. Mass spectrometer : Triple TOF 5600 Gradient : Two Gradient Programs, 4 hrs & 8 hrs

Reference: Hydrophilic Interaction Chromatography Using a Meter-Scale Monolithic silica capillary Column for Proteomics LC-MS, K Horie et al. Anal. Chem. 2014, 86, 3817-3824

Performance of MonoCap HILIC-UP HighResolution 2000

Conditions Column : MonoCap HILIC-UP High Resolution 2000 Eluent : A) CH3CN : H2O=10/90 (0.5% CH3COOH) B) CH3CN : H2O=95/5 (0.5% CH3COOH) A/B=0/100-(240 min)-20/80-(10 min)-100/0-(10 min)-100/0 Flow Rate : 0.5 μL Injection Vol. : 1 μL (1 mg/mL) Detection : TIC MS (m/z 300-1500) Sample : Tryptic Digest of Hela Cell Lysate, 5 ug

Number of Identified Peptides : 7,194 (14736 PSM*) Number of Identified Proteins : 2,201

*Peptide Spectrum Match

Total Ion Chromatogram Base Peak Chromatogram

20000000 40000000 60000000 80000000 100000000 120000000 50 100 150 200 250 500000 1000000 1500000 2000000 2500000 3000000 3500000 4000000 50 100 150 200 250

Time (min) Intensity Intensity

Reference: Results of MonoCap C18 HighResolution 2000 Number of Identified Peptides: 8,358 Number of Identified Proteins: 1,992 Gradient Program: 4 hrs

Time (min)

Ordering Information

Description Model Remarks Cat.No. Zero Dead Volume Union U-435 For 1/16“ SUS Tubing 6010-72352

Column Connection

Purchase the following connection kit when directly connecting the column to the injector of a nano-LC instrument.

Description Contents Cat.No. MonoCap C18 HighResolution 2000 Connection Kit 1/16“ PEEK Ferrule, SUS Nut, Sleeve, 2 pcs each. 1/32“ PEEK Ferrule, SUS Nut, Sleeve, 2 pcs each. 5020-10017

MonoCap C18 HighResolution 2000 Connection Kit

1/16“ PEEK Ferrule 1/32“ PEEK Ferrule 1/16“ PEEK Sleeve 1/32“ PEEK Sleeve 1/32“ SUS Nut 1/16“ SUS Nut

Example of Column Connection

Pump Eluent Injector MonoCap C18 HighResolution 2000 LC-MS/MS

Installing MonoCap to the appropriate End-fittings

• 1. Prepare either 1/16” or 1/32” end-fittings and sleeve included in the MonoCap

connection kit (Cat# 5020-10017). Ferrule Nut PEEK Sleeve

Installing MonoCap to the appropriate End-fittings

• 2. Insert the sleeve into a nut and ferrule. Then insert a fused silica capillary tube into

the sleeve. Ferrule Nut PEEK Sleeve Fused Silica Capillary Tube (To be prepared by the customer)

Installing MonoCap to the appropriate End-fittings

• 3. Insert the pre-assembled fitting into the injection port. Then tighten the nut by two
• r three turns using a wrench to make certain that the ferrule grabs the sleeve.

Wrench Nut PEEK Sleeve Fused Silica Capillary Tube (To be prepared by the customer)

Installing MonoCap to the appropriate End-fittings

• 4. Now the ferrule is fixed to the sleeve. Remove the fused silica capillary tube from the

sleeve. The ferrule is now fixed to the sleeve Nut PEEK Sleeve Fused Silica Capillary Tube (To be prepared by the customer)

Installing MonoCap to the appropriate End-fittings

• 5. Insert the MonoCap C18 HighResolution 2000 column into the sleeve. Then push the

sleeve all the way into the port for a zero dead volume connection that the assembly seats firmly. Finally, keep pushing the MonCap C18 column into the port direction and tighten the nut by two or three turns. Nut PEEK Sleeve MonoCap C18 HighResolution 2000

Installing MonoCap to the appropriate End-fittings

• 6. Gently pull the MonoCap C18 column to the direction shown below in blue. Make

sure the column is not loose and it is firmly installed. MonoCap C18 HighResolution 2000

Column Connection

Purchase the following union when using a tubing that generates any indirect connection to the column.

Description Model Remarks Cat.No. Zero Dead Volume Union U-435 For 1/16“ SUS Tubing 6010-72352

Example of Column Connection using nanoViper

Pump Eluent Injector MonoCap C18 HighResolution 2000 LC-MS/MS

Union nanoViper

Installing MonoCap to nanoViper

• 1. Prepare 1/16” end-fittings and sleeve included in the MonoCap connection kit

(Cat# 5020-10017). Ferrule Nut PEEK Sleeve

Installing MonoCap to nanoViper

• 2. Insert the sleeve into a nut and ferrule. Then insert a fused silica capillary tube into

the sleeve. Ferrule Nut PEEK Sleeve Fused Silica Capillary Tube (To be prepared by the customer)

Installing MonoCap to nanoViper

• 3. Insert the pre-assembled fitting into the injection port. Then tighten the nut by two
• r three turns using a wrench to make certain that the ferrule grabs the sleeve.

Wrench Nut PEEK Sleeve Fused Silica Capillary Tube (To be prepared by the customer)

Installing MonoCap to nanoViper

• 4. Now the ferrule is fixed to the sleeve. Remove the fused silica capillary tube from the

sleeve. The ferrule is now fixed to the sleeve Nut PEEK Sleeve Fused Silica Capillary Tube (To be prepared by the customer)

Installing MonoCap to nanoViper

Union nanoViper

• 5. Connect the MonoCap C18 HighResolution 2000 column into union. Push the sleeve

all the way into the union for a zero dead volume connection that the assembly seats

• firmly. Keep pushing the MonCap C18 column into the union and tighten the nut by

two or three turns. Gently pull the MonoCap C18 to confirm if the column is not loose and it is firmly installed. Finally, fingertight the nanoViper to the injection port. PEEK Sleeve MonoCap C18 HighResolution 2000

Installing MonoCap to nanoViper

Example of Column Connection

Pump Eluent Injector MonoCap C18 HighResolution 2000 LC-MS/MS

ここにnanoViperをかまして 接続する方法の写真を挿入 Union Sprayer Sleeve MonoCap 18