Supporting Information Mobility of Proteins in Porous Substrates under ...
Supporting Information Mobility of Proteins in Porous Substrates under Electrospray Ionization Conditions Bin Hu†§, Zhong-Ping Yao†‡§* †
State Key Laboratory for Chirosciences, Food Safety and Technology Research Centre and Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong SAR, China ‡
Key Laboratory of Natural Resources of Changbai Mountain and Functional Molecules (Yanbian University), Ministry of Education, Yanji, 133002, China §
State Key Laboratory of Chinese Medicine and Molecular Pharmacology (Incubation) and Shenzhen Key Laboratory of Food Biological Safety Control, Shenzhen Research Institute of The Hong Kong Polytechnic University, Shenzhen, 518057, China
*Corresponding author: Dr. Zhong-Ping Yao Department of Applied Biology and Chemical Technology The Hong Kong Polytechnic University Hung Hom, Kowloon Hong Kong Tel.: +852-3400 8792 Fax: +852-2364 9932 Email: [email protected]
Contents: Experimental Section Figures S1-S11
S-1
Experimental Section Materials: The proteins, chemicals and organic solvents used in this study were purchased from Sigma (St. Louis, MO, USA). Peptides were purchased from Bachem Bionscience Inc (PA, USA). Water was Milli-Q water generated from a Synthesis A10 Milli-Q water generator (Millipore, Billerica, MA, USA). Polyester tips and polyethylene tips were purchased from Deqing Huihua Pen Co., Ltd. (Deqing, China). Wooden tips (wooden toothpicks) and fresh porcine heart were purchased from ParknShop (Hong Kong). Dense polyethylene tips were obtained by pressing the tips for 12 hours, and the densities were obtained by their weights divided by their volumes calculated based on the measurement data. The mixture of insulin and alpha-lactalbumin was prepared in methanol/water/formic acid (1/1/0.1%, v/v/v), with 4 µM for alpha-lactalbumin and 1.5 µM for insulin in the solutions. Different concentrations of the proteins in each mixture were used to generate similar intensities of the peaks for comparison. The mixture of tripeptides was prepared in methanol/water/formic acid (1/1/0.1%, v/v/v) with 5×10-5 M for each peptide. The cytochrome c solution was 5×10-4 M in acetonitrile/water/acetic acid (25/75/0.1, v/v/v) and myoglobin 20 µM in 12.5% acetonitrile. These peptide and protein solutions were prepared with concentrations similar to those in the relevant literatures. All porous tips are washed twice with methanol/water (1:1, v/v) in Eppendorf tube (1.5 ml) with vortex for 1 minute (~ 1500 rpm) and then dried in air before use or reuse. Extraction of proteins from porcine heart: a small piece (~10 mg) of fresh porcine heart was soaked with 1.5 µL methanol/acetone (1/1, v/v) and vortexed (~1500 rpm) for 3 min. The extract was then used for the analysis. Extraction of proteins remained on the porous tips: After the spray ionization was completed, the tips were cut into two parts at the position that was 1.0 mm from the sharp end. Each part was soaked with 50 µL solvent and vortexed for 1 min. The extracts were analyzed using conventional ESI-MS. Methanol/water/formic acid (1/1/0.1% v/v/v) was used as the extraction solvent, except that 20 mM ammonium acetate was used for the myoglobin study. Mass spectrometry: Mass spectra were acquired on a Q-TOF2 quadrupole time-of-flight mass spectrometer (Micromass, Manchester, UK). The high voltage and cone voltage were set at 3.5 kV and 30 V, respectively. The ion source temperature was set at 80ºC, and the scan time was 0.2 sec. Porous-tip ESI: As shown in Figure S1, porous tips (tip-end size: ~0.2 mm, length: 1.5 cm) were connected to the high voltage supply of the mass spectrometer via a metal clip. The tips were parallel to the mass spectrometer inlet with the sharp end ~ 8.0 mm away from the inlet. Typically, aliquots of 10 µL sample solutions were loaded onto the tips at a position about 5 mm from the sharp end. For the experiments with continuously supplied solvents, solvents were introduced onto the tips by a syringe pump (KD Scientific Inc., Holliston, MA, USA) with a flow rate of 2 µL/min. Other settings were similar to those previously described for the wooden-tip ESI (see: Hu, B.; So,
S-2
P. K.; Chen, H. W.; Yao, Z. P. Anal Chem 2011, 83, 8201-8207. Hu, B.; So, P. K.; Yao, Z. P. J Am Soc Mass Spectrom 2013, 24, 57-65.). Conventional ESI: Sample solutions were introduced to a capillary via a transfer line by a syringe pump (KD Scientific Inc., Holliston, MA, USA). The flow rate of the sample was 5 µL/min. Nitrogen was used as the sheath gas, cone gas and desolvation gas with a flow rate of 15 L/h, 75 L/h and 250 L/h, respectively. SEM analysis: all SEM images were obtained with a JSM-6490 equipment (JEOL Ltd. Tokyo, Japan).
S-3
Figure S1. Solution movement on porous substrates under ESI conditions. At stage 1, bulk solution movement is mainly involved and proteins of different sizes are sprayed out together. At stage 2, migration inside the microchannels is the main form of solution movement and proteins with higher mobility are easier to be sprayed out of the porous tips to be detected.
S-4
a)
b)
Figure S2. SICs of the 4+ ion of insulin and the 9+ ion of alpha-lactalbumin obtained by analysis of the alpha-lactalbumin/insulin sample using polyester-tip ESI-MS a) with the same tip after cleaning and reuse, and b) with different tips.
S-5
Figure S3. Detection of the proteins remained on the tip after polyester-tip ESI-MS analysis of the alpha-lactalbumin/insulin sample. a) Spectrum obtained by analysis of the front 1.0 mm part of the polyester tip. b) Spectrum obtained by analysis of the other part of the polyester tip.
S-6
Figure S4. SEM images of the polyester tips: a) magnifications of 30 for the surface; b) magnifications of 500 for the surface; c) magnifications of 150 for the cross section; and d) magnifications of 1100 for the cross section
S-7
a)
insulin (+4)
alpha-lactalbumin (+9)
b)
insulin (+4)
alpha-lactalbumin (+9)
c) insulin (+4)
alpha-lactalbumin (+9)
Figure S5. Analysis of the alpha-lactalbumin/insulin sample using porous-tip ESI-MS with a) wooden tip, b) polyethylene tip (ρ = 0.60 g/cm3), and c) polyethylene tip (ρ = 1.03 g/cm3).
S-8
b)
a) H N H2 N
O C N H
O
O C
H N
OH H2N
N H
O
O
H2N
O
N H
O
C-terminal amino acid Nonpolar surface area (Å2)* Glycine(G) 47 Alanine(A) 86 Valine (V) 135 Leucine (L) 164 Phenylalaine (F) 194 Tyrosine (Y) 152 *Ref: Eur. J. Med. Chem. 1983, 18, 369
OH O
Gly-Gly-Val (GGL, MW 231)
Gly-Gly-Ala (GGA, MW 203)
Gly-Gly-Gly (GGG, MW 189)
O C
H N
OH
OH
H N O
N H
H2N
O
Gly-Gly-Leu (GGL, MW 245) 100
c)
N H
O
O
O
2.35 0
1
2.452
3
2.55 4 time / min
52.65
e) T2
2.75 7
6
100
GGG
80
Relative response
T1
Relative abundance
Relative abundance
GGG GGA GGV GGL GGY GGF
Polyester
d)
GGG GGA GGV GGY GGL GGF
80 60
20 20
O
Gly-Gly-Tyr(GGY, MW 295)
T2
60 40 40
OH N H
H2N
Gly-Gly-Phe (GGF, MW 279)
T1
O C
H N
OH
100 80
00
f)
O C
H N
OH
60
GGV
80
GGY
60
GGL
40
GGF
20
Polyester
y = -0.5903x + 116.81 R² = 0.9661
0 5.25
h) 100
5.75 Time (min)
6.25
30
6.75
Wood 60 40
50
70 90 110 130 150 170 190 210 Non-polar surface Area (Å2)
i)
14
GGG GGA GGV GGY GGL GGF
80
12 SIC area
4.75
Relative response
40 20
0
Wood 10 8
20
y = -0.0303x + 14.789 R² = 0.8667
6
0 2.35
2.45
2.55 Time (min)
100 100
2.65
30
2.75
300
Relative response
Polyethylene
GGG GGV GGA
6060
GGY GGV GGL
4040
GGL
GGF
GGY
2020
0.47
70 90 110 130 150 170 190 210 Non-polar surface Area (Å2)
Polyethylene Polyethylene
200 150 100
y = -1.3395x + 313.41 R² = 0.9506
50
GGF
00 4.75
50
k)
250
GGG GGA
8080
SIC area
j)
g)
100
GGA
Polyester
SIC area
H2N
O C
0 0.97
5.25
1.47
1.97
5.75
2.47
6.25
2.97
3.47
Time (min)
6.75
3.97
30
50
70 90 110 130 150 170 190 210 Non-polar surface Area (Å2)
Figure S6. Analysis of the tripeptide mixture using porous-tip ESI-MS. a) Structures of the six tripeptides. b) Nonpolar surface areas of the C-terminal residues. c) SICs of the six tripeptides obtained by using polyester-tip ESI-MS. d) and e) mass spectra for durations T1 and T2 in c). f), h) and j) Normalized SICs of the six tripeptides at stage 2 obtained by using wooden tips, polyester tips and polyethylene tips (ρ = 0.60 g/cm3), respectively. g), i) and k) Plots of normalized SIC area against nonpolar surface area of the six tripeptides for analysis with wooden tips, polyester tips and polyethylene tips (ρ = 0.60 g/cm3), respectively.
S-9
Relative abundance
100
Area for scan i: Ai = 0.2
80
Ii-1 + Ii 2 n
SIC area: A = ∑Ai
60
i=1
40 20 0 4.75 t0
Number of scans: n = Ii-1
tn – t0 0.2
Ii
0.2 s
5.25
5.75 Time (sec)
6.25
6.75 tn
Figure S7. Calculation of the SIC area. The SIC area was obtained by summation of SIC area for each scan, which was calculated by scan time (0.2 sec) multiplied by mean relative abundance of the scan. Ii-1 and Ii are the relative abundance at scan i-1 and i, respectively, and t0 and tn are the start time and end time of stage 2, respectively.
S-10
a) TIC
+7 +17
b)
TIC
+7 +17
c)
TIC
+7 +17
Figure S8. Analysis of cytochrome c using porous-tip ESI-MS with a) wooden tip, b) polyethylene tip (ρ = 0.60 g/cm3), and c) polyethylene tip (ρ =1.03 g/cm3).
S-11
a)
+14 +9
+8 ACN: 25 % +7
b) ACN: 20 %
c) ACN: 15 %
d)
ACN: 10 %
e) ACN: 5 %
Figure S9. Spectra obtained by using conventional ESI-MS for analysis of cytochrome c (10 µM) in different compositions of acetonitrile (ACN) in water containing 0.1% acetic acid: a) 25% ACN; b) 20% ACN; c) 15% ACN; d) 10% ACN; e) 5% ACN.
S-12
a)
+14 +8
+9
1.0 µM +7
b)
10.0 µM
c)
100.0 µM
Figure S10. Spectra obtained by using conventional ESI-MS for analysis of cytochrome c with different concentrations in acetonitrile/water/acetic acetate (25/75/0.1, v/v/v): a) 1.0 µM; b) 10.0 µM; c) 100.0 µM.
S-13
a)
Heme
[PC(34:1)+Na]+ [PC(34:1)+K]+
+15α +14α +13α
+12α
+11α +10α
+9 β
+8 β
+8 β
b)
+7 β +9α +9 β +11α +10α +10 β
c)
+14α
Figure S11. Analysis of the crude extract of fresh porcine heart. a) Spectrum obtained by direct analysis using conventional ESI-MS. Lipids, and heme, α-chain and β-chain of hemeglobin were observed. The spectrum was similar to what we previously obtained by direct ionization mass spectrometric analysis of fresh porcine heart with methanol/acetone (1/1, v/v) as the solvent (Ref: Analyst, 2012, 137, 3613-1639). b) Spectrum obtained for the extract of the front 1.0 mm part of the polyester tip after the polyester-tip ESI-MS analysis. c) Spectrum obtained for the extract of the other part of the polyester tip after the polyester-tip ESI-MS analysis. The presented data showed that β-chain of hemeglobin was predominant in the front 1.0 mm part of the tip, while almost no β-chain and mainly heme and α-chain of hemeglobin were observed in the remainder part of the tip, demonstrating the preparative separation of the technique to the crude extract.
S-14
State Key Laboratory for Chirosciences, Food Safety and Technology Research Centre and Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong SAR, China ‡
Key Laboratory of Natural Resources of Changbai Mountain and Functional Molecules (Yanbian University), Ministry of Education, Yanji, 133002, China §
State Key Laboratory of Chinese Medicine and Molecular Pharmacology (Incubation) and Shenzhen Key Laboratory of Food Biological Safety Control, Shenzhen Research Institute of The Hong Kong Polytechnic University, Shenzhen, 518057, China
*Corresponding author: Dr. Zhong-Ping Yao Department of Applied Biology and Chemical Technology The Hong Kong Polytechnic University Hung Hom, Kowloon Hong Kong Tel.: +852-3400 8792 Fax: +852-2364 9932 Email: [email protected]
Contents: Experimental Section Figures S1-S11
S-1
Experimental Section Materials: The proteins, chemicals and organic solvents used in this study were purchased from Sigma (St. Louis, MO, USA). Peptides were purchased from Bachem Bionscience Inc (PA, USA). Water was Milli-Q water generated from a Synthesis A10 Milli-Q water generator (Millipore, Billerica, MA, USA). Polyester tips and polyethylene tips were purchased from Deqing Huihua Pen Co., Ltd. (Deqing, China). Wooden tips (wooden toothpicks) and fresh porcine heart were purchased from ParknShop (Hong Kong). Dense polyethylene tips were obtained by pressing the tips for 12 hours, and the densities were obtained by their weights divided by their volumes calculated based on the measurement data. The mixture of insulin and alpha-lactalbumin was prepared in methanol/water/formic acid (1/1/0.1%, v/v/v), with 4 µM for alpha-lactalbumin and 1.5 µM for insulin in the solutions. Different concentrations of the proteins in each mixture were used to generate similar intensities of the peaks for comparison. The mixture of tripeptides was prepared in methanol/water/formic acid (1/1/0.1%, v/v/v) with 5×10-5 M for each peptide. The cytochrome c solution was 5×10-4 M in acetonitrile/water/acetic acid (25/75/0.1, v/v/v) and myoglobin 20 µM in 12.5% acetonitrile. These peptide and protein solutions were prepared with concentrations similar to those in the relevant literatures. All porous tips are washed twice with methanol/water (1:1, v/v) in Eppendorf tube (1.5 ml) with vortex for 1 minute (~ 1500 rpm) and then dried in air before use or reuse. Extraction of proteins from porcine heart: a small piece (~10 mg) of fresh porcine heart was soaked with 1.5 µL methanol/acetone (1/1, v/v) and vortexed (~1500 rpm) for 3 min. The extract was then used for the analysis. Extraction of proteins remained on the porous tips: After the spray ionization was completed, the tips were cut into two parts at the position that was 1.0 mm from the sharp end. Each part was soaked with 50 µL solvent and vortexed for 1 min. The extracts were analyzed using conventional ESI-MS. Methanol/water/formic acid (1/1/0.1% v/v/v) was used as the extraction solvent, except that 20 mM ammonium acetate was used for the myoglobin study. Mass spectrometry: Mass spectra were acquired on a Q-TOF2 quadrupole time-of-flight mass spectrometer (Micromass, Manchester, UK). The high voltage and cone voltage were set at 3.5 kV and 30 V, respectively. The ion source temperature was set at 80ºC, and the scan time was 0.2 sec. Porous-tip ESI: As shown in Figure S1, porous tips (tip-end size: ~0.2 mm, length: 1.5 cm) were connected to the high voltage supply of the mass spectrometer via a metal clip. The tips were parallel to the mass spectrometer inlet with the sharp end ~ 8.0 mm away from the inlet. Typically, aliquots of 10 µL sample solutions were loaded onto the tips at a position about 5 mm from the sharp end. For the experiments with continuously supplied solvents, solvents were introduced onto the tips by a syringe pump (KD Scientific Inc., Holliston, MA, USA) with a flow rate of 2 µL/min. Other settings were similar to those previously described for the wooden-tip ESI (see: Hu, B.; So,
S-2
P. K.; Chen, H. W.; Yao, Z. P. Anal Chem 2011, 83, 8201-8207. Hu, B.; So, P. K.; Yao, Z. P. J Am Soc Mass Spectrom 2013, 24, 57-65.). Conventional ESI: Sample solutions were introduced to a capillary via a transfer line by a syringe pump (KD Scientific Inc., Holliston, MA, USA). The flow rate of the sample was 5 µL/min. Nitrogen was used as the sheath gas, cone gas and desolvation gas with a flow rate of 15 L/h, 75 L/h and 250 L/h, respectively. SEM analysis: all SEM images were obtained with a JSM-6490 equipment (JEOL Ltd. Tokyo, Japan).
S-3
Figure S1. Solution movement on porous substrates under ESI conditions. At stage 1, bulk solution movement is mainly involved and proteins of different sizes are sprayed out together. At stage 2, migration inside the microchannels is the main form of solution movement and proteins with higher mobility are easier to be sprayed out of the porous tips to be detected.
S-4
a)
b)
Figure S2. SICs of the 4+ ion of insulin and the 9+ ion of alpha-lactalbumin obtained by analysis of the alpha-lactalbumin/insulin sample using polyester-tip ESI-MS a) with the same tip after cleaning and reuse, and b) with different tips.
S-5
Figure S3. Detection of the proteins remained on the tip after polyester-tip ESI-MS analysis of the alpha-lactalbumin/insulin sample. a) Spectrum obtained by analysis of the front 1.0 mm part of the polyester tip. b) Spectrum obtained by analysis of the other part of the polyester tip.
S-6
Figure S4. SEM images of the polyester tips: a) magnifications of 30 for the surface; b) magnifications of 500 for the surface; c) magnifications of 150 for the cross section; and d) magnifications of 1100 for the cross section
S-7
a)
insulin (+4)
alpha-lactalbumin (+9)
b)
insulin (+4)
alpha-lactalbumin (+9)
c) insulin (+4)
alpha-lactalbumin (+9)
Figure S5. Analysis of the alpha-lactalbumin/insulin sample using porous-tip ESI-MS with a) wooden tip, b) polyethylene tip (ρ = 0.60 g/cm3), and c) polyethylene tip (ρ = 1.03 g/cm3).
S-8
b)
a) H N H2 N
O C N H
O
O C
H N
OH H2N
N H
O
O
H2N
O
N H
O
C-terminal amino acid Nonpolar surface area (Å2)* Glycine(G) 47 Alanine(A) 86 Valine (V) 135 Leucine (L) 164 Phenylalaine (F) 194 Tyrosine (Y) 152 *Ref: Eur. J. Med. Chem. 1983, 18, 369
OH O
Gly-Gly-Val (GGL, MW 231)
Gly-Gly-Ala (GGA, MW 203)
Gly-Gly-Gly (GGG, MW 189)
O C
H N
OH
OH
H N O
N H
H2N
O
Gly-Gly-Leu (GGL, MW 245) 100
c)
N H
O
O
O
2.35 0
1
2.452
3
2.55 4 time / min
52.65
e) T2
2.75 7
6
100
GGG
80
Relative response
T1
Relative abundance
Relative abundance
GGG GGA GGV GGL GGY GGF
Polyester
d)
GGG GGA GGV GGY GGL GGF
80 60
20 20
O
Gly-Gly-Tyr(GGY, MW 295)
T2
60 40 40
OH N H
H2N
Gly-Gly-Phe (GGF, MW 279)
T1
O C
H N
OH
100 80
00
f)
O C
H N
OH
60
GGV
80
GGY
60
GGL
40
GGF
20
Polyester
y = -0.5903x + 116.81 R² = 0.9661
0 5.25
h) 100
5.75 Time (min)
6.25
30
6.75
Wood 60 40
50
70 90 110 130 150 170 190 210 Non-polar surface Area (Å2)
i)
14
GGG GGA GGV GGY GGL GGF
80
12 SIC area
4.75
Relative response
40 20
0
Wood 10 8
20
y = -0.0303x + 14.789 R² = 0.8667
6
0 2.35
2.45
2.55 Time (min)
100 100
2.65
30
2.75
300
Relative response
Polyethylene
GGG GGV GGA
6060
GGY GGV GGL
4040
GGL
GGF
GGY
2020
0.47
70 90 110 130 150 170 190 210 Non-polar surface Area (Å2)
Polyethylene Polyethylene
200 150 100
y = -1.3395x + 313.41 R² = 0.9506
50
GGF
00 4.75
50
k)
250
GGG GGA
8080
SIC area
j)
g)
100
GGA
Polyester
SIC area
H2N
O C
0 0.97
5.25
1.47
1.97
5.75
2.47
6.25
2.97
3.47
Time (min)
6.75
3.97
30
50
70 90 110 130 150 170 190 210 Non-polar surface Area (Å2)
Figure S6. Analysis of the tripeptide mixture using porous-tip ESI-MS. a) Structures of the six tripeptides. b) Nonpolar surface areas of the C-terminal residues. c) SICs of the six tripeptides obtained by using polyester-tip ESI-MS. d) and e) mass spectra for durations T1 and T2 in c). f), h) and j) Normalized SICs of the six tripeptides at stage 2 obtained by using wooden tips, polyester tips and polyethylene tips (ρ = 0.60 g/cm3), respectively. g), i) and k) Plots of normalized SIC area against nonpolar surface area of the six tripeptides for analysis with wooden tips, polyester tips and polyethylene tips (ρ = 0.60 g/cm3), respectively.
S-9
Relative abundance
100
Area for scan i: Ai = 0.2
80
Ii-1 + Ii 2 n
SIC area: A = ∑Ai
60
i=1
40 20 0 4.75 t0
Number of scans: n = Ii-1
tn – t0 0.2
Ii
0.2 s
5.25
5.75 Time (sec)
6.25
6.75 tn
Figure S7. Calculation of the SIC area. The SIC area was obtained by summation of SIC area for each scan, which was calculated by scan time (0.2 sec) multiplied by mean relative abundance of the scan. Ii-1 and Ii are the relative abundance at scan i-1 and i, respectively, and t0 and tn are the start time and end time of stage 2, respectively.
S-10
a) TIC
+7 +17
b)
TIC
+7 +17
c)
TIC
+7 +17
Figure S8. Analysis of cytochrome c using porous-tip ESI-MS with a) wooden tip, b) polyethylene tip (ρ = 0.60 g/cm3), and c) polyethylene tip (ρ =1.03 g/cm3).
S-11
a)
+14 +9
+8 ACN: 25 % +7
b) ACN: 20 %
c) ACN: 15 %
d)
ACN: 10 %
e) ACN: 5 %
Figure S9. Spectra obtained by using conventional ESI-MS for analysis of cytochrome c (10 µM) in different compositions of acetonitrile (ACN) in water containing 0.1% acetic acid: a) 25% ACN; b) 20% ACN; c) 15% ACN; d) 10% ACN; e) 5% ACN.
S-12
a)
+14 +8
+9
1.0 µM +7
b)
10.0 µM
c)
100.0 µM
Figure S10. Spectra obtained by using conventional ESI-MS for analysis of cytochrome c with different concentrations in acetonitrile/water/acetic acetate (25/75/0.1, v/v/v): a) 1.0 µM; b) 10.0 µM; c) 100.0 µM.
S-13
a)
Heme
[PC(34:1)+Na]+ [PC(34:1)+K]+
+15α +14α +13α
+12α
+11α +10α
+9 β
+8 β
+8 β
b)
+7 β +9α +9 β +11α +10α +10 β
c)
+14α
Figure S11. Analysis of the crude extract of fresh porcine heart. a) Spectrum obtained by direct analysis using conventional ESI-MS. Lipids, and heme, α-chain and β-chain of hemeglobin were observed. The spectrum was similar to what we previously obtained by direct ionization mass spectrometric analysis of fresh porcine heart with methanol/acetone (1/1, v/v) as the solvent (Ref: Analyst, 2012, 137, 3613-1639). b) Spectrum obtained for the extract of the front 1.0 mm part of the polyester tip after the polyester-tip ESI-MS analysis. c) Spectrum obtained for the extract of the other part of the polyester tip after the polyester-tip ESI-MS analysis. The presented data showed that β-chain of hemeglobin was predominant in the front 1.0 mm part of the tip, while almost no β-chain and mainly heme and α-chain of hemeglobin were observed in the remainder part of the tip, demonstrating the preparative separation of the technique to the crude extract.
S-14
