Do we still need nanospray for exploratory proteomics? · Proteins Peptides PSMs. 2.1 x 100 mm; 300...

Do we still need nanospray for exploratory proteomics?

PharmDr. Juraj Lenčo, Ph.D.Department of Analytical Chemistry

Faculty of Pharmacy in Hradec KrálovéCharles University

LC-MS installations

nanoLC/MS

Background

Q Exactive PlusnanoLC/nanoESI

conv. flow LC/HESI-II

Identical sample

Identical gradient

Identical DDA method

Identical evaluation

Methods

HeLa cells

Preview TM

Search parameters

Spectra processing

MaxQuant

ProteomeDiscoverer

Mascot

Main DDA search

LC peaks parameters

Andromeda

Percolator

DDA of HeLa rLys-C/trypsin digest

MS2 PSMs/peptides Explained MS2 [%]

Proteins Peptides PSMs

Benchmark nanoLC0.075 x 250 mm; 250 nL/min

Acclaim™ PepMap™ C18, 100Å, 2 μm

2 g , T o p 6

2 g , T o p 1 2

n a n o 3 7 5 n g

P ro t e in s P e p tid e s P S M s

2 g , T o p 6

2 g , T o p 1 2

n a n o 3 7 5 n g

M S 2 P S M / p e p t id e s E x p la in e d M S 2 [% ]

9 3 8 6 .5 k 8 .0 k

1 6 .7 k 1 .2 3 4 7 .6

2381 15.9k 23.5k

38.6k 1.48 61.0



2.1 x 100 mm; 300 µL/min

Poroshell 120Å C18 2.7 μm

Narrow-bore LC

2 g , T o p 6

2 g , T o p 1 2

n a n o L C


2 g , T o p 6

2 g , T o p 1 2

n a n o L C


9 3 8 6 .5 k 8 .0 k

1 6 .7 k 1 .2 3 4 7 .6

2381 15.9k 23.5k

38.6k 1.48 61.0



0521 12.8k 23.4k

24.4k 1.22 14.0

DDA settings Top12 Top6

Fill time [ms] 50 110

DDA optimization

2.1 x 100 mm; 300 µL/min


Narrow-bore LC

2 g , T o p 6

2 g , T o p 1 2

n a n o L C


2 g , T o p 6

2 g , T o p 1 2

n a n o L C


9 3 8 6 .5 k 8 .0 k

1 6 .7 k 1 .2 3 4 7 .6

2381 15.9k 23.5k

38.6k 1.48 61.0



1.0 x 100 mm; 68 µL/min


Microbore LC

2.1 mm 1.0 mm

2.12

1.02= 4.41

O p t im , T o p 1 2 , 1 .0 m m

O p t im , T o p 6 , 1 .0 m m

T o p 6 , 1 .0 m m

T o p 6 , 2 .1 m m

n a n o L C


O p t im , T o p 1 2 , 1 .0 m m

O p t im , T o p 6 , 1 .0 m m

T o p 6 , 1 .0 m m

T o p 6 , 2 .1 m m

n a n o L C


8 .5 k

1 4 .4 k

1 1 0 7

1 448

1 619

6 .8 k

9 .8 k

1 2 .0 k 1 9 .3 k

1 8 .3 k

1 9 .7 k

3 3 .1 k

1 .2 6

1 .46

1 .61

4 6 .7

7 2.9

5 8.4

1.0 x 100 mm; 68 µL/min

Microbore LC



2381 15.9k 23.5k

38.6k 1.48 61.0

𝜎𝑐𝑜𝑙2 =

𝑉02

𝑁∗∙ (1 + 𝑘𝑒)

2

𝑘𝑒 =𝑘0

𝑏 ∙ 𝑘0 + 1

𝑘0 =1

𝑏𝑒𝑏∙𝑘𝑔 − 1

𝑘𝑔 =1

1.15 ∙ 𝑏 𝑏 = 𝑆 ∙ 𝛥𝛷 ∙𝑉0

𝑡𝑔 ∙ 𝐹

𝑁∗ = 5.54𝐺 ∙ 𝑡0 1 + 𝑘𝑒

𝑊0.5

2

𝐺 =1 + 𝑝 +

𝑝2

3

1 + 𝑝

𝑝 =2.3 ∙ 𝑘0 ∙ 𝑏

𝑘0 + 1

𝜎𝑒𝑐2 = 𝜎𝑖𝑛𝑗

2 + 𝜎𝑝𝑟𝑒2 + 𝜎𝑝𝑜𝑠𝑡

2 + 𝜎𝑑𝑒𝑡2 + 𝜎𝑜𝑡ℎ𝑒𝑟

2

𝜎𝑡𝑢𝑏𝑖𝑛𝑔2 =

π2 ∙ 𝑟4 ∙ 𝐿2

3 + 24 ∙ 𝜋 ∙ 𝐿 ∙𝐷𝑚𝐹

𝐷𝑚 =8.341 ∙ 10−8 ∙ 𝑇

𝜂 ∙13 𝑀𝑤

𝜂 = 2.414 ∙ 10−5 ∙ 10247.8𝑇−140

𝜎𝑖𝑛𝑗2 = 𝑘𝑖𝑛𝑗 ∙

𝑉𝑖𝑛𝑗2

12

𝜎𝑑𝑒𝑡2 = 𝑘𝑐𝑒𝑙𝑙 ∙

𝑉𝑐𝑒𝑙𝑙2

12+ 𝜏2 ∙ 𝐹2

𝑬𝒓 = 𝟏𝟎𝟎 ∙𝝈𝒄𝒐𝒍𝟐

𝝈𝒄𝒐𝒍𝟐 + 𝝈𝒆𝒄

𝟐

Microbore LC

0 .0 1 0 .1 1 1 0 1 0 0

0

5 0

1 0 0

2e c [ L

2]

Er

[%]

4 .6 m m

1 .0 m m

2 .1 m m

9 0

3.5 μL281 μL2

E r[%

]

σ2ec [μL2]

0.18 μL2

RT [min]

𝐸𝑟 = 100 ∙𝜎𝑐𝑜𝑙2

𝜎𝑐𝑜𝑙2 + 𝜎𝑒𝑐

2

Microbore LC

0 1 0 0 2 0 0 3 0 0 4 0 0

1

1 0

1 0 0

F [ L / m in ]

2

ec [

L2

]

O rig in a l c o n f ig .

In it ia lly o p t . c o n f ig .

F in a lly o p t . c o n f ig .

3.5 μL2

2.2 μL2

42 μL2

σ2

ec[μ

L2]

F [μL/min]

0 .0 1 0 .1 1 1 0 1 0 0

0

5 0

1 0 0

2e c [ L

2]

Er

[%]

4 .6 m m

1 .0 m m

2 .1 m m

9 0

3.5 μL281 μL2

E r[%

]

σ2ec [μL2]

0.18 μL2

Microbore LC

s 0 5

s 0 6

s 0 2 1 1 .4 s

2 .4 s

2 .4 s

Origi. config

Finally opt. config.

RT [min]

Intensity fold change

W50% [s]

RT [min]

Finally opt. config.Narrow-bore LC

Microbore LC

O p t im , T o p 1 2 , 1 .0 m m

O p t im , T o p 6 , 1 .0 m m

T o p 6 , 1 .0 m m

T o p 6 , 2 .1 m m

n a n o L C


O p t im , T o p 1 2 , 1 .0 m m

O p t im , T o p 6 , 1 .0 m m

T o p 6 , 1 .0 m m

T o p 6 , 2 .1 m m

n a n o L C


1 4 .4 k1 4 4 8

1 619

9 .8 k

1 2 .0 k 1 9 .3 k

1 9 .7 k

3 3 .1 k

1 .4 6

1 .61

7 2 .9

5 8.4

1.0 x 100 mm; 68 µl/min

Microbore LC



2381 15.9k 23.5k

38.6k 1.48 61.0

O p t im , T o p 1 2 , 1 .0 m m

O p t im , T o p 6 , 1 .0 m m

T o p 6 , 1 .0 m m

T o p 6 , 2 .1 m m

n a n o L C


O p t im , T o p 1 2 , 1 .0 m m

O p t im , T o p 6 , 1 .0 m m

T o p 6 , 1 .0 m m

T o p 6 , 2 .1 m m

n a n o L C


1 6 1 9 1 2 .0 k 1 9 .3 k

3 3 .1 k 1 .6 1 5 8 .4

1.0 x 100 mm; 68 µl/min

Microbore LC



15.9k 23.5k

38.6k 1.48 61.0

2381

100°C

80°C

60°C

40°C

η pressure

Dm

Dm

longitudinal diffusion

resistance to mass transfer

second. interactions kinetics

Pro isomerization kinetics

carry-over

Temperature

O p t im , T o p 1 2 , 7 0 °C

O p t im , T o p 1 2 , 5 5 °C

O p t im , T o p 1 2 , 4 0 °C

O p t im , T o p 1 2 , R T

n a n o L C


O p t im , T o p 1 2 , 7 0 °C

O p t im , T o p 1 2 , 5 5 °C

O p t im , T o p 1 2 , 4 0 °C

O p t im , T o p 1 2 , R T

n a n o L C


2 0 .8 k1 9 9 0

1 8 9 6

1 5 .0 k

1 4 .2 k 2 0 .7 k

3 5 .9 k

3 6 .1 k

1 .3 8

1 .4 6

5 7 .9

5 7 .3



2381 15.9k 23.5k

38.6k 1.48 61.0

Temperature

A C D E F G H I K L M N P Q R S T V W Y

R T 5 0 °C 6 0 °C 7 0 °C 8 0 °C

G IF P V L C K D P V Q E A W A E D V D L

G IF P V L C K D

R T 5 0 °C 6 0 °C 7 0 °C 8 0 °C

Q E Y D E S G P S IV H R

E S G P S IV H R

Last AAs in semitryptic peptides

GIFPVLCKDPVQEAWAEDVDLGIFPVLCKD

QEYDESGPSIVHRESGPSIVHR

Peptide degradation test

Columnlength [mm] Pc

100 234

250 283

RT [min]

Halo Peptide 160Å C18 2.7 μm



2 5 c m

1 0 c m

n a n o L C


2 5 c m

1 0 c m

n a n o L C


2 2 .8 k2 2 3 4 1 7 .7 k

3 6 .8 k 1 .2 9 6 2 .1

1 5 .9 k2381 23.5k

38.6k 1.48 61.0

Columnlength [mm]

0 .1 % F A

0 .4 % H A c

0 .5 % H A c

0 .1 % F A

n a n o L C


0 .1 % F A

0 .4 % H A c

0 .5 % H A c

0 .1 % F A

n a n o L C


2 3 2 3 1 8 .6 k 2 4 .2 k

3 7 .0 k 1 .3 0 6 5 .3

2 3 .7 k



Acidifiers

2381 15.9k 23.5k

38.6k 1.48 61.0

Additivesfor reduction of charge states

2+

3+

4+

5+

0

2 5

5 0

7 5

C h a rg e

PS

Ms

[%

]

nanoLCmicrobore LC

with DMSOPSM

[%

]

1 > 1

0

2 5

5 0

7 5

1 0 0

Charge states per peptide

Charge

3 % D M S O

0 .1 % F A

3 % D M S O

n o D M S O

n a n o L C


3 % D M S O

0 .1 % F A

3 % D M S O

n o D M S O

n a n o L C


2 2 3 4

2 4 .9 k2 8 3 5

2 7 9 7

2 1 .7 k

2 1 .4 k 2 4 .5 k

3 7 .9 k

3 7 .5 k

1 .1 5

1 .1 4

6 5 .6

6 5 .3



15.9k 23.5k

38.6k 1.48 61.0

Additivesfor reduction of charge states

D M S O

0 .1 % F A

n o D M S O

C o n v e n t . L C (2 g )

n a n o L C (3 7 5 n g )

D M S O

0 .1 % F A

n o D M S O

C o n v e n t . L C (2 g )

n a n o L C (3 7 5 n g )


2 2 3 4 1 7 .7 k

2 4 .9 k

2 4 .9 k

2 2 .8 k

3 6 .8 k 1 . 2 9 6 2 . 1

2 8 3 5

2 8 3 5

2 7 9 7

2 1 .7 k

2 1 .7 k

2 1 .4 k 2 4 .5 k

3 7 .9 k

3 7 .9 k

3 7 .5 k

1 . 1 5

1 . 1 5

1 . 1 4

6 5 . 6

6 5 . 6

6 5 . 3



Do we still need nanospray for exploratory proteomics?

This work was supported by the STARSS project (Reg. No. CZ.02.1.01/0.0/0.0/15_003/0000465) co-funded by ERDF.

0.075 x 250 mm250 nL/min

22 °C0.1% FA

1.0 x 250 mm68 μL/min

55 °C3% DMSO, 0.1/0.4% FA/Hac

𝜎𝑒𝑐2

Conventional-flow LC-MS analyses for proteomic

applications

Label-free MS1 quantification

Control1000 ng bacterialin 2000 ng background

Sample A1000 ngin 2000 ng

Sample B316 ngin 2000 ng

Sample C100 ngin 2000 ng

2 0 4 0 6 0

-6

-3

0

3

6

9

R T [ m in ]

R

T [

s]

C o n t r o l S a m p le B S a m p le CΔ

RT

[s]

RT [min] for Sample A





- 6 - 4 - 2 0 2

0

2 5 0

5 0 0

- 6 - 4 - 2 0 2

0

2 5 0

5 0 0

- 6 - 4 - 2 0 2

0

4 0 0

8 0 0Median

0.99Median

0.31Median

0.11





Phosphopeptides

0 k 2 k 4 k

5 0 0 g

1 5 8 g

5 0 g

P h o sp h o p e p t id e s

(p h o s p h o R S IP 0 .9 5 )

0 .0 k 2 .5 k 5 .0 k


0 .0 k 2 .5 k 5 .0 k

T o ta l p e p t id e s

3 .0 k 4 .3 k 5 .0 k

TiO2

IMAC

Combined

0 k 2 k 4 k

5 0 0 g

1 5 8 g

5 0 g


(p h o s p h o R S IP 0 .9 5 )

0 .0 k 2 .5 k 5 .0 k


0 .0 k 2 .5 k 5 .0 k


1 .4 k 2 .3 k 2 .7 k

0 k 2 k 4 k

5 0 0 g

1 5 8 g

5 0 g


(p h o s p h o R S IP 0 .9 5 )

0 .0 k 2 .5 k 5 .0 k


0 .0 k 2 .5 k 5 .0 k


2 .5 k 3 .5 k 4 .0 k

https://pubs.acs.org/doi/10.1021/acs.analchem.8b00525

Do we still need nanospray for exploratory proteomics? · Proteins Peptides PSMs. 2.1 x 100 mm; 300...

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Transcript of Do we still need nanospray for exploratory proteomics? · Proteins Peptides PSMs. 2.1 x 100 mm; 300...