monocap highresolution 2000 series - gl sciences · monocap highresolution 2000 series optimized...

MonoCap HighResolution 2000 Series

Optimized for Identification of Peptides /Proteinsfor 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 2000Silica : High Purity Monolithic Silica GelThrough-pore Size : 2 μmMesopore Size : 15 nmBonded Phase : Octadecyl GroupsEnd-capping : YesInternal Diameter : 0.1 mm I.D.Length : 2000 mmRecommended Flow Rate Ranges : 0.3 μL/min ～ 1 μL/minMaximum Operating Pressure : 35 MPa, 300 Bar

Physical Properties of MonoCap HighResolution 2000

MonoCap HILIC-UP HighResolution 2000Silica : High Purity Monolithic Silica GelThrough-pore Size : 2 μmMesopore Size : 12 nmBonded Phase : Ureidopropyl GroupsEnd-capping : NoneInternal Diameter : 0.1 mm I.D.Length : 2000 mmRecommended Flow Rate Ranges : 0.3 μL/min ～ 1 μL/minMaximum Operating Pressure : 35 MPa, 300 BarRemarks : 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 Moreabove factors

MonoCap HighResolution2000 mm columns

Traditional Particle Packed150 mm columns

Performance of MonoCap C18 HighResolution 2000

Column NameNumber of

Identified Proteins in average

Analysis Time

MonoCap C18 HighResolution 20000.1 mm I.D. x 2000 mm

2,087 (2013, 2116, 2131) 10 Hours

Particle packed column0.1 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


180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600160140120100806040200

[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)

ConditionsSystem 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.DetectionSample

● ● ● 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 weightConditionsSystem 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.DetectionSample

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 600160140120100806040200

180 200 220 240 260 280 300 320 340 360160140120100806040200


Time (min)


Time (min)


Time (min)

● ● ● are analytes having the same molecular weightColumn : 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/minEluent : A) CH3CN (0.1%HCOOH), B) H2O (0.1%HCOOH)

A / B = 10 / 90 - 180, 300, 600 min- 45/55Sample : Tryptic digest of proteinsInjection vol. : 5 μL


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 ptsLTQ-OrbitrapXL: cut off=0.05

All data were acquired, provided by Astellas Pharma Inc., Mr. Hiramoto


MonoCap C18 12hr gradient

LTQ-XL(Top5 CID)

MonoCap C18 6hr gradient

LTQ-XL(Top5 CID)

15 cm Particle packed2hr gradient

LTQ-OrbitrapXL(Top3 CID)

Offgel fractionator(12 frs)

15cm packed 2hrOrbitrapXL(Top3 CID)

Rat brain microsomeTryptic digestca.25 μg

1,772 1,661 915 2,800

Rat brain microsomeTryptic digestiTRAQ5 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 2000Comparison of Number of Identified Proteins


RT: 0.00 - 119.98

0 10 20 30 40 50 60 70 80 90 100 110Time (min)

0

20

40

60

80

100

Rel

ative

Abu

ndan

ce

60.80621.6653

68.17588.3217

98.59416.3369

72.60644.3710 99.68

444.368076.91

923.506854.15

535.309796.48

581.3677

91.58816.4934

45.97612.2755

77.35599.3589

100.85415.211641.66

560.778031.53451.7808

84.38951.8036

12.76437.8893

105.10639.6283

0.18445.1200

18.59445.1200

NL:1.75E8Base Peak MS FMBM06_1H19_01

RT: 0.00 - 720.02

0 50 100 150 200 250 300 350 400 450 500 550 600 650 700Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

387.38766.51

367.42705.56

451.80852.40420.82

675.53350.08567.38

271.54622.08

191.38535.58

255.57579.37

638.10403.16501.49

817.06330.59588.57237.52

709.52 288.92652.57

463.58952.06

185.11598.18

505.79746.26

314.57607.68

485.90713.60

617.25430.34

137.24612.59

171.10470.58

130.21516.25

597.39416.55

220.07553.71

76.46452.14

29.49438.42 524.14

848.27704.42445.11

690.20445.13

NL:7.59E6Base Peak M S monollith2m_110902_06

RT: 0.00 - 360.01

0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

152.34567.35

165.79766.50 178.50

924.20

189.57956.83

279.04822.64128.20

622.13150.51

588.63138.27652.57 212.24

817.04124.67579.51 285.89

563.40194.99952.12103.86

535.55 116.18709.54 224.15

581.54 259.91416.51

83.75612.54

74.82561.05

61.55452.09

250.42449.96

50.53471.05

287.57656.82

235.22515.97

40.16455.47

335.36445.02

6.15444.96

354.51444.94

319.68445.02

NL:1.26E7Base Peak M S monollith2m_110901_04

0.1 x 150 mm 3 μm particle packed column210 protein hit (OrbitrapXL, HCDCID top3)

Rat brain microsome 5 μg tryptic digest iTRAQ

0.1 x 2000 mm MonoCap C18 40 ℃ 6hr gradient1,187 protein hit(LTQ-XL CID top3）

0.1 x 2000 mm MonoCap C18 40 ℃ 12 hr gradient1,482 protein hit(LTQ-XL CID top3）


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-OrbitapXLInjecting two times separately)

Purificatin of phosphopeptides via HAMMOC Method(Phosphoric acid, Ammonia, Pyrrolidine gradual elution)

Anti P-Tyr Ab

ElutePass 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

267phosphopeptides

2D LC

MonoCap 2m

1/21/2

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 5,5004,000

HILIC-UP C18

Column Size : 2000 mm x 0.1 mm I.D. Mass spectrometer : Triple TOF 5600Gradient : 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

ConditionsColumn : MonoCap HILIC-UP High Resolution 2000Eluent : 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 μLInjection 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

0

20000000

40000000

60000000

80000000

100000000

120000000

0 50 100 150 200 2500

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

0 50 100 150 200 250

Time (min)

Inte

nsity

Inte

nsity

Reference:Results of MonoCap C18 HighResolution 2000

Number of Identified Peptides: 8,358Number of Identified Proteins: 1,992Gradient 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 ConnectionPurchase 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 Ferrule1/16“ PEEK Sleeve 1/32“ PEEK Sleeve

1/32“ SUS Nut1/16“ SUS Nut

Example of Column Connection

Pump

Eluent

Injector MonoCap C18HighResolution 2000 LC-MS/MS

Installing MonoCap to the appropriate End-fittings1. 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-fittings2. 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 thecustomer)

Installing MonoCap to the appropriate End-fittings3. Insert the pre-assembled fitting into the injection port. Then tighten the nut by two

or three turns using a wrench to make certain that the ferrule grabs the sleeve.

Wrench

Nut

PEEK Sleeve


Installing MonoCap to the appropriate End-fittings4. 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


Installing MonoCap to the appropriate End-fittings5. 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 C18HighResolution 2000

Installing MonoCap to the appropriate End-fittings6. 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.


Column ConnectionPurchase 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


UnionnanoViper

Installing MonoCap to nanoViper1. 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



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

Wrench

Nut

PEEK Sleeve



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



UnionnanoViper

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



Example of Column Connection

Pump

Eluent


ここにnanoViperをかまして接続する方法の写真を挿入

Union Sprayer

SleeveMonoCap 18

monocap highresolution 2000 series - gl sciences · monocap highresolution 2000 series optimized...

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