Lecture Notes - Mid-semester examination (50 marks; 2 hours)

: Lecture Notes - Mid-semester examination (50 marks; 2 hours)

Created by: Rohnit Roy
 Page 1


N NP PT TE EL L  W WE EB B  C CO OU UR RS SE E  – –  A AD DV VA AN NC CE ED D  C CL LI IN NI IC CA AL L  P PR RO OT TE EO OM MI IC CS S                                                                                                                            
  
Mid-semester examination (50 marks; 2 hours) 
 
Question 1. Explain the following (pointed and short answers) (10 marks)   
 
1.1. Minimum labeling vs. Saturation labeling   
 
1.2. IPG strips vs. tube gels 
 
1.3. Forward vs. reverse proteomics   
 
1.4. On-gel electrophoresis vs. Off-gel electrophoresis 
 
1.5. Ion exchange vs. gel exclusion chromatography   
  
1.6. Soft vs. hard ionization 
 
1.7. Equilibration I vs. II      
 
1.8. “b” vs. “y” ions 
 
1.9. Proteomics vs. protein chemistry     
 
1.10. Passive rehydration vs. Active rehydration 
 
 
PROF. SANJEEVA SRIVASTAVA 
DEPARTMENT OF BIOSCIENCES & BIOENGINEERING 
 IIT BOMBAY, MUMBAI, INDIA Page 1 
Page 2


N NP PT TE EL L  W WE EB B  C CO OU UR RS SE E  – –  A AD DV VA AN NC CE ED D  C CL LI IN NI IC CA AL L  P PR RO OT TE EO OM MI IC CS S                                                                                                                            
  
Mid-semester examination (50 marks; 2 hours) 
 
Question 1. Explain the following (pointed and short answers) (10 marks)   
 
1.1. Minimum labeling vs. Saturation labeling   
 
1.2. IPG strips vs. tube gels 
 
1.3. Forward vs. reverse proteomics   
 
1.4. On-gel electrophoresis vs. Off-gel electrophoresis 
 
1.5. Ion exchange vs. gel exclusion chromatography   
  
1.6. Soft vs. hard ionization 
 
1.7. Equilibration I vs. II      
 
1.8. “b” vs. “y” ions 
 
1.9. Proteomics vs. protein chemistry     
 
1.10. Passive rehydration vs. Active rehydration 
 
 
PROF. SANJEEVA SRIVASTAVA 
DEPARTMENT OF BIOSCIENCES & BIOENGINEERING 
 IIT BOMBAY, MUMBAI, INDIA Page 1 
N NP PT TE EL L  W WE EB B  C CO OU UR RS SE E  – –  A AD DV VA AN NC CE ED D  C CL LI IN NI IC CA AL L  P PR RO OT TE EO OM MI IC CS S                                                                                                                            
  
Question 2. Identify the problem as demonstrated in these 2D gels and describe your suggestions 
to overcome the problem in each gel image (1.5+1 = 2.5 X 4 = 10 marks)   
 
 2D gel image Problem (s)   Your recommendation (how to 
overcome the problem) 
i 
 
  
 
 
 
 
ii 
 
 
  
 
 
 
 
 
 
 
 
iii 
 
 
  
 
 
 
 
 
 
 
 
iv 
 
   
 
 
 
 
 
 
 
 
 
PROF. SANJEEVA SRIVASTAVA 
DEPARTMENT OF BIOSCIENCES & BIOENGINEERING 
 IIT BOMBAY, MUMBAI, INDIA Page 2 
Page 3


N NP PT TE EL L  W WE EB B  C CO OU UR RS SE E  – –  A AD DV VA AN NC CE ED D  C CL LI IN NI IC CA AL L  P PR RO OT TE EO OM MI IC CS S                                                                                                                            
  
Mid-semester examination (50 marks; 2 hours) 
 
Question 1. Explain the following (pointed and short answers) (10 marks)   
 
1.1. Minimum labeling vs. Saturation labeling   
 
1.2. IPG strips vs. tube gels 
 
1.3. Forward vs. reverse proteomics   
 
1.4. On-gel electrophoresis vs. Off-gel electrophoresis 
 
1.5. Ion exchange vs. gel exclusion chromatography   
  
1.6. Soft vs. hard ionization 
 
1.7. Equilibration I vs. II      
 
1.8. “b” vs. “y” ions 
 
1.9. Proteomics vs. protein chemistry     
 
1.10. Passive rehydration vs. Active rehydration 
 
 
PROF. SANJEEVA SRIVASTAVA 
DEPARTMENT OF BIOSCIENCES & BIOENGINEERING 
 IIT BOMBAY, MUMBAI, INDIA Page 1 
N NP PT TE EL L  W WE EB B  C CO OU UR RS SE E  – –  A AD DV VA AN NC CE ED D  C CL LI IN NI IC CA AL L  P PR RO OT TE EO OM MI IC CS S                                                                                                                            
  
Question 2. Identify the problem as demonstrated in these 2D gels and describe your suggestions 
to overcome the problem in each gel image (1.5+1 = 2.5 X 4 = 10 marks)   
 
 2D gel image Problem (s)   Your recommendation (how to 
overcome the problem) 
i 
 
  
 
 
 
 
ii 
 
 
  
 
 
 
 
 
 
 
 
iii 
 
 
  
 
 
 
 
 
 
 
 
iv 
 
   
 
 
 
 
 
 
 
 
 
PROF. SANJEEVA SRIVASTAVA 
DEPARTMENT OF BIOSCIENCES & BIOENGINEERING 
 IIT BOMBAY, MUMBAI, INDIA Page 2 
N NP PT TE EL L  W WE EB B  C CO OU UR RS SE E  – –  A AD DV VA AN NC CE ED D  C CL LI IN NI IC CA AL L  P PR RO OT TE EO OM MI IC CS S                                                                                                                            
  
Question 3. (2 X 6 = 12 marks)   
Glutathione peroxidase 3 (Gpx3) is an important antioxidant enzyme in Saccharomyces 
cerevisiae. A study compared the wild type S. cerevisiae strain with its Gpx3 mutant strain by 
using two dimensional gel electrophoresis on 4-7 pI, 24 cm IPG strips. 2D gel images were 
analyzed using software and 6 spots were selected for further electrospray ionization quadrupole 
time-of-flight tandem mass spectrometry (ESI-Q-TOF MS/MS) analysis. Raw MS data was 
processed by MASCOT search program (Version 2.2, Matrix Science) and used for protein 
identification. Details of proteins identified by MS/MS is listed in table.  
Analyse this data in Table and determine what will be your evaluation for data (proteins A-F) on 
a scale of 1-5 (1 – Best data; 2 – Good data; 3 – OK but acceptable; 4 – Doubtful, there is further 
need to look at raw data; 5 – Reject). Please fill in your option (only one option for each) in 
given space. Also, write-down your rationale for choosing which option is correct.  
 
Protei
n 
Species pI Accession 
No. 
Peptid
e 
match 
MOWS
E score 
Fold 
chang
e 
Your answer 
(1-5 scale) 
A Saccharomyces 
pombe 
5.9 gi|6715442 1 13 -3.1  
B Saccharomyces 
cerevisiae 
6.3 gi|195650293 13 200 +2.3  
C Saccharomyces 
cerevisiae 
7.0 gi|195639750 7 25 +1.7  
D Bacillus subtilis  5.5 gi|3036946 1 17 +2.6  
E Saccharomyces 
cerevisiae 
8.6 gi|13096169 2 39 -2.6  
F Saccharomyces 
cerevisiae 
5.1 gi|194701222 3 60 -1.9  
 
  
 
 
PROF. SANJEEVA SRIVASTAVA 
DEPARTMENT OF BIOSCIENCES & BIOENGINEERING 
 IIT BOMBAY, MUMBAI, INDIA Page 3 
Page 4


N NP PT TE EL L  W WE EB B  C CO OU UR RS SE E  – –  A AD DV VA AN NC CE ED D  C CL LI IN NI IC CA AL L  P PR RO OT TE EO OM MI IC CS S                                                                                                                            
  
Mid-semester examination (50 marks; 2 hours) 
 
Question 1. Explain the following (pointed and short answers) (10 marks)   
 
1.1. Minimum labeling vs. Saturation labeling   
 
1.2. IPG strips vs. tube gels 
 
1.3. Forward vs. reverse proteomics   
 
1.4. On-gel electrophoresis vs. Off-gel electrophoresis 
 
1.5. Ion exchange vs. gel exclusion chromatography   
  
1.6. Soft vs. hard ionization 
 
1.7. Equilibration I vs. II      
 
1.8. “b” vs. “y” ions 
 
1.9. Proteomics vs. protein chemistry     
 
1.10. Passive rehydration vs. Active rehydration 
 
 
PROF. SANJEEVA SRIVASTAVA 
DEPARTMENT OF BIOSCIENCES & BIOENGINEERING 
 IIT BOMBAY, MUMBAI, INDIA Page 1 
N NP PT TE EL L  W WE EB B  C CO OU UR RS SE E  – –  A AD DV VA AN NC CE ED D  C CL LI IN NI IC CA AL L  P PR RO OT TE EO OM MI IC CS S                                                                                                                            
  
Question 2. Identify the problem as demonstrated in these 2D gels and describe your suggestions 
to overcome the problem in each gel image (1.5+1 = 2.5 X 4 = 10 marks)   
 
 2D gel image Problem (s)   Your recommendation (how to 
overcome the problem) 
i 
 
  
 
 
 
 
ii 
 
 
  
 
 
 
 
 
 
 
 
iii 
 
 
  
 
 
 
 
 
 
 
 
iv 
 
   
 
 
 
 
 
 
 
 
 
PROF. SANJEEVA SRIVASTAVA 
DEPARTMENT OF BIOSCIENCES & BIOENGINEERING 
 IIT BOMBAY, MUMBAI, INDIA Page 2 
N NP PT TE EL L  W WE EB B  C CO OU UR RS SE E  – –  A AD DV VA AN NC CE ED D  C CL LI IN NI IC CA AL L  P PR RO OT TE EO OM MI IC CS S                                                                                                                            
  
Question 3. (2 X 6 = 12 marks)   
Glutathione peroxidase 3 (Gpx3) is an important antioxidant enzyme in Saccharomyces 
cerevisiae. A study compared the wild type S. cerevisiae strain with its Gpx3 mutant strain by 
using two dimensional gel electrophoresis on 4-7 pI, 24 cm IPG strips. 2D gel images were 
analyzed using software and 6 spots were selected for further electrospray ionization quadrupole 
time-of-flight tandem mass spectrometry (ESI-Q-TOF MS/MS) analysis. Raw MS data was 
processed by MASCOT search program (Version 2.2, Matrix Science) and used for protein 
identification. Details of proteins identified by MS/MS is listed in table.  
Analyse this data in Table and determine what will be your evaluation for data (proteins A-F) on 
a scale of 1-5 (1 – Best data; 2 – Good data; 3 – OK but acceptable; 4 – Doubtful, there is further 
need to look at raw data; 5 – Reject). Please fill in your option (only one option for each) in 
given space. Also, write-down your rationale for choosing which option is correct.  
 
Protei
n 
Species pI Accession 
No. 
Peptid
e 
match 
MOWS
E score 
Fold 
chang
e 
Your answer 
(1-5 scale) 
A Saccharomyces 
pombe 
5.9 gi|6715442 1 13 -3.1  
B Saccharomyces 
cerevisiae 
6.3 gi|195650293 13 200 +2.3  
C Saccharomyces 
cerevisiae 
7.0 gi|195639750 7 25 +1.7  
D Bacillus subtilis  5.5 gi|3036946 1 17 +2.6  
E Saccharomyces 
cerevisiae 
8.6 gi|13096169 2 39 -2.6  
F Saccharomyces 
cerevisiae 
5.1 gi|194701222 3 60 -1.9  
 
  
 
 
PROF. SANJEEVA SRIVASTAVA 
DEPARTMENT OF BIOSCIENCES & BIOENGINEERING 
 IIT BOMBAY, MUMBAI, INDIA Page 3 
N NP PT TE EL L  W WE EB B  C CO OU UR RS SE E  – –  A AD DV VA AN NC CE ED D  C CL LI IN NI IC CA AL L  P PR RO OT TE EO OM MI IC CS S                                                                                                                            
  
Question 4. In MS spectrum provided to you, please write-down the peptide sequence and also 
show your steps in answer book. You are free to use calculator (8 marks). 
 
 
 
 
 
 
 
 
 
PROF. SANJEEVA SRIVASTAVA 
DEPARTMENT OF BIOSCIENCES & BIOENGINEERING 
 IIT BOMBAY, MUMBAI, INDIA Page 4 
Page 5


N NP PT TE EL L  W WE EB B  C CO OU UR RS SE E  – –  A AD DV VA AN NC CE ED D  C CL LI IN NI IC CA AL L  P PR RO OT TE EO OM MI IC CS S                                                                                                                            
  
Mid-semester examination (50 marks; 2 hours) 
 
Question 1. Explain the following (pointed and short answers) (10 marks)   
 
1.1. Minimum labeling vs. Saturation labeling   
 
1.2. IPG strips vs. tube gels 
 
1.3. Forward vs. reverse proteomics   
 
1.4. On-gel electrophoresis vs. Off-gel electrophoresis 
 
1.5. Ion exchange vs. gel exclusion chromatography   
  
1.6. Soft vs. hard ionization 
 
1.7. Equilibration I vs. II      
 
1.8. “b” vs. “y” ions 
 
1.9. Proteomics vs. protein chemistry     
 
1.10. Passive rehydration vs. Active rehydration 
 
 
PROF. SANJEEVA SRIVASTAVA 
DEPARTMENT OF BIOSCIENCES & BIOENGINEERING 
 IIT BOMBAY, MUMBAI, INDIA Page 1 
N NP PT TE EL L  W WE EB B  C CO OU UR RS SE E  – –  A AD DV VA AN NC CE ED D  C CL LI IN NI IC CA AL L  P PR RO OT TE EO OM MI IC CS S                                                                                                                            
  
Question 2. Identify the problem as demonstrated in these 2D gels and describe your suggestions 
to overcome the problem in each gel image (1.5+1 = 2.5 X 4 = 10 marks)   
 
 2D gel image Problem (s)   Your recommendation (how to 
overcome the problem) 
i 
 
  
 
 
 
 
ii 
 
 
  
 
 
 
 
 
 
 
 
iii 
 
 
  
 
 
 
 
 
 
 
 
iv 
 
   
 
 
 
 
 
 
 
 
 
PROF. SANJEEVA SRIVASTAVA 
DEPARTMENT OF BIOSCIENCES & BIOENGINEERING 
 IIT BOMBAY, MUMBAI, INDIA Page 2 
N NP PT TE EL L  W WE EB B  C CO OU UR RS SE E  – –  A AD DV VA AN NC CE ED D  C CL LI IN NI IC CA AL L  P PR RO OT TE EO OM MI IC CS S                                                                                                                            
  
Question 3. (2 X 6 = 12 marks)   
Glutathione peroxidase 3 (Gpx3) is an important antioxidant enzyme in Saccharomyces 
cerevisiae. A study compared the wild type S. cerevisiae strain with its Gpx3 mutant strain by 
using two dimensional gel electrophoresis on 4-7 pI, 24 cm IPG strips. 2D gel images were 
analyzed using software and 6 spots were selected for further electrospray ionization quadrupole 
time-of-flight tandem mass spectrometry (ESI-Q-TOF MS/MS) analysis. Raw MS data was 
processed by MASCOT search program (Version 2.2, Matrix Science) and used for protein 
identification. Details of proteins identified by MS/MS is listed in table.  
Analyse this data in Table and determine what will be your evaluation for data (proteins A-F) on 
a scale of 1-5 (1 – Best data; 2 – Good data; 3 – OK but acceptable; 4 – Doubtful, there is further 
need to look at raw data; 5 – Reject). Please fill in your option (only one option for each) in 
given space. Also, write-down your rationale for choosing which option is correct.  
 
Protei
n 
Species pI Accession 
No. 
Peptid
e 
match 
MOWS
E score 
Fold 
chang
e 
Your answer 
(1-5 scale) 
A Saccharomyces 
pombe 
5.9 gi|6715442 1 13 -3.1  
B Saccharomyces 
cerevisiae 
6.3 gi|195650293 13 200 +2.3  
C Saccharomyces 
cerevisiae 
7.0 gi|195639750 7 25 +1.7  
D Bacillus subtilis  5.5 gi|3036946 1 17 +2.6  
E Saccharomyces 
cerevisiae 
8.6 gi|13096169 2 39 -2.6  
F Saccharomyces 
cerevisiae 
5.1 gi|194701222 3 60 -1.9  
 
  
 
 
PROF. SANJEEVA SRIVASTAVA 
DEPARTMENT OF BIOSCIENCES & BIOENGINEERING 
 IIT BOMBAY, MUMBAI, INDIA Page 3 
N NP PT TE EL L  W WE EB B  C CO OU UR RS SE E  – –  A AD DV VA AN NC CE ED D  C CL LI IN NI IC CA AL L  P PR RO OT TE EO OM MI IC CS S                                                                                                                            
  
Question 4. In MS spectrum provided to you, please write-down the peptide sequence and also 
show your steps in answer book. You are free to use calculator (8 marks). 
 
 
 
 
 
 
 
 
 
PROF. SANJEEVA SRIVASTAVA 
DEPARTMENT OF BIOSCIENCES & BIOENGINEERING 
 IIT BOMBAY, MUMBAI, INDIA Page 4 
N NP PT TE EL L  W WE EB B  C CO OU UR RS SE E  – –  A AD DV VA AN NC CE ED D  C CL LI IN NI IC CA AL L  P PR RO OT TE EO OM MI IC CS S                                                                                                                            
  
Question 5. Please write the answer of following questions (2X5 = 10 marks)  
5.1. In reverse phase chromatography what are mobile and stationary phases?  
5.2. What is the extraction solution used for in-gel digestion procedure?  
5.3. The processes by which an ion is selected for MS/MS analysis is known as…………? 
5.4. In multidimensional separation, size exclusion chromatography followed by reversed phase 
is less used due to_____________ 
5.5. The reflector in TOF analyzer is used for which purpose?  
 
PROF. SANJEEVA SRIVASTAVA 
DEPARTMENT OF BIOSCIENCES & BIOENGINEERING 
 IIT BOMBAY, MUMBAI, INDIA Page 5 
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