Proteomics Problem Set
Answers
( A)
Human plasma is notoriously difficult for proteomics analysis because of high dynamic range. A variety of pre-fractionation approaches are used to
deal with this, such as immunoaffinity isolation of proteins of interest (using immobolized monoclonal antibodies), or, the opposite, immunodepletion (i.e.
removal of the most abundant proteins, such as albumin and globulins. Other pre-fractionation approaches can be used, as well.
(B)
4.8 nM correspons to 80 µg/L of myoglobin. Assuming 100% recovery, 1.25 mL of plasma should be enough for visualization of myoglobin using
Coumassie blue stain, and ~63 µL – in the case of Silver stain. Given the total plasma protein concentration of ~ 70 mg/mL, these volumes of plasma
correspond to 88 mg and 4.4 mg of total protein. In the latter case, such amount can me loaded onto micropreparative Immobiline gel strips. In the former
case, though, IEF device with higher protein load would be necessary (e.g., the membrane system shown on the lecture slides).
2)
Because of sample complexity, several separation steps are used in proteomics studies, coupled online and off-line. Examples of coupled 2dimensional separations can be found in the figure below:
Marjorie L. Fournier; Joshua M. Gilmore; Skylar A. Martin-Brown; Michael P. Washburn; Chem. Rev. 2007, 107, 3654-3686
Rough prefractionation methods can be added to these schemes, in order to increase resolution and recovery of proteins with “extreme” physico-chemical
properties (differential solubilisation, narrow-range IPG strips) or recovery of low-abundant proteins (preparative IEF, organelle prefractionation, affinity
isolation).
Combinations of separation steps will be less efficient if the 2 methods based on similar protein or peptide properties are chosen, e.g. IEF and ion exchange
and capillary zone electrophoresis, SDS-page and gel-permeation chromatography, etc. It is worth to remember that mass spectrometer is a mass- (mass-tocharge) based separation tool, so any mass-based separation straight before the MS analysis will be less useful than the other separation methods.
Enzymatic digestion can be performed either before the 2D-separation, or between the two steps of separation.
3 (A) Using NCBI protein database ( http://www.ncbi.nlm.nih.gov/protein/ ) and MS-Digest tool (Protein Prospector), perform in-silico digest for the following
proteins: murine myoglobin, human myoglobin, chicken ovalbumin, bovine serum albumin, human fibrinogen. Consider oxidation of methionin as a variable
modification.
(B)
Compare the in-silico digests of the two myoglobins. Which peptides are necessary in order to distinguish these proteins by their PMF spectra?
>gi|44955888|ref|NP_976312.1| myoglobin [Homo sapiens]
Considered modifications: | Oxidation (M) |
Digest Used: Trypsin
Max. # Missed Cleavages: 0
User AA Formula 1: C2 H3 N1 O1
Minimum Digest Fragment Mass: 500
Maximum Digest Fragment Mass: 4000
Minimum Digest Fragment Length: 5
Index Number: 1
pI of Protein: 7.1
Protein MW: 17184
Amino Acid Composition: A12 C1 D8 E14 F7 G15 H9 I8 K20 L17 M4 N3 P5 Q7 R2 S7 T4 V7 W2 Y2
MGLSDGEWQLVLNVWGKVEADIPGHGQEVLIRLFKGHPETLEKFDKFKHLKSEDEMKASEDLKKHGATVL
TALGGILKKKGHHEAEIKPLAQSHATKHKIPVKYLEFISECIIQVLQSKHPGDFGADAQGAMNKALELFR
KDMASNYKELGFQG
Number
1
1
1
1
1
1
1
1
m/z(mi)
650.3144
662.3355
738.2974
748.4352
754.2924
828.3556
844.3505
910.4629
m/z(av)
650.7133
662.7217
738.7960
748.9057
754.7953
828.9245
844.9239
911.0083
Modifications Start
149
58
52
135
1Oxidation
52
142
1Oxidation
142
36
End
154
63
57
140
57
148
148
43
MissedCleavages
Sequence
0
(K)ELGFQG(-)
0
(K)ASEDLK(K)
0
(K)SEDEMK(A)
0
(K)ALELFR(K)
0
(K)SEDEMK(A)
0
(K)DMASNYK(E)
0
(K)DMASNYK(E)
0
(K)GHPETLEK(F)
1
1
1
1
1
1
1
1
1350.8104
1515.6645
1531.6594
1632.8704
1853.9617
1913.0089
1931.9684
1947.9633
1351.6420
1516.6420
1532.6414
1633.8560
1855.0761
1914.2853
1933.2503
1949.2496
1Oxidation
1Oxidation
65
120
120
18
81
104
1
1
78
134
134
32
97
119
17
17
0
0
0
0
0
0
0
0
(K)HGATVLTALGGILK(K)
(K)HPGDFGADAQGAMNK(A)
(K)HPGDFGADAQGAMNK(A)
(K)VEADIPGHGQEVLIR(L)
(K)GHHEAEIKPLAQSHATK(H)
(K)YLEFISECIIQVLQSK(H)
(-)MGLSDGEWQLVLNVWGK(V)
(-)MGLSDGEWQLVLNVWGK(V)
>gi|255708425|ref|NP_001157520.1| myoglobin [Mus musculus]
MGLSDGEWQLVLNVWGKVEADLAGHGQEVLIGLFKTHPETLDKFDKFKNLKSEEDMKGSEDLKKHGCTVL
TALGTILKKKGQHAAEIQPLAQSHATKHKIPVKYLEFISEIIIEVLKKRHSGDFGADAQGAMSKALELFR
NDIAAKYKELGFQG
pI of Protein: 7.1
Protein MW: 17070
Amino Acid Composition: A13 C1 D9 E13 F7 G15 H7 I9 K20 L18 M3 N3 P3 Q7 R2 S7 T6 V7 W2 Y2
Number
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
m/z(mi)
631.3410
648.3199
650.3144
738.2974
748.4352
754.2924
940.4734
1426.8086
1478.6329
1494.6278
1708.9772
1786.9195
1896.0225
1931.9684
1947.9633
m/z(av)
631.7106
648.6947
650.7133
738.7960
748.9057
754.7953
941.0347
1427.7599
1479.5774
1495.5768
1710.0765
1787.9850
1897.1949
1933.2503
1949.2496
Modifications Start
141
58
149
52
135
1Oxidation
52
36
65
120
1Oxidation
120
104
81
18
1
1Oxidation
1
End
146
63
154
57
140
57
43
78
134
134
117
97
35
17
17
Missed Cleavages
Sequence
0
(R)NDIAAK(Y)
0
(K)GSEDLK(K)
0
(K)ELGFQG(-)
0
(K)SEEDMK(G)
0
(K)ALELFR(N)
0
(K)SEEDMK(G)
0
(K)THPETLDK(F)
0
(K)HGCTVLTALGTILK(K)
0
(R)HSGDFGADAQGAMSK(A)
0
(R)HSGDFGADAQGAMSK(A)
0
(K)YLEFISEIIIEVLK(K)
0
(K)GQHAAEIQPLAQSHATK(H)
0
(K)VEADLAGHGQEVLIGLFK(T)
0
(-)MGLSDGEWQLVLNVWGK(V)
0
(-)MGLSDGEWQLVLNVWGK(V)
As can be seen, the sequences of the 2 myoglobins, human (H) and murine (M) are quite similar. Yet, their peptide mass fingerprint spectra in the MW ranfe
from 500 to 2000 Da are quite distinct, with only 2 common peptides, the first and the last ones in the sequence (not considering the Met-oxidized forms).
Interestingly, the 52-57 fragments of both proteins are different, yet isobaric, as the two AA’s, D and E, are swopped. This difference will be seen in the
MS/MS spectra of the peptides.
M
H
1 mglsdgewql vlnvwgkvea dlaghgqevl iglfkthpet ldkfdkfknl kseedmkgse
1 mglsdgewql vlnvwgkvea dipghgqevl irlfkghpet lekfdkfkhl ksedemkase
M
H
61 dlkkhgctvl talgtilkkk gqhaaeiqpl aqshatkhki pvkylefise iiievlkkrh
61 dlkkhgatvl talggilkkk ghheaeikpl aqshatkhki pvkylefise ciiqvlqskh
M
H
121 sgdfgadaqg amskalelfr ndiaakykel gfqg
121 pgdfgadaqg amnkalelfr kdmasnykel gfqg
4 (A) Well, nowadays both ionization methods can be equally useful for PMF. MALDI yields almost exclusively singly-charged species, which makes PMF
spectra interpretation easier, yet now the data analysis is usually performed with the use of search tools which consider multiply-charged ions. ESI-ion trap
instruments, widely used in the proteomics facilities, cannot provide high mass accuracy which would allow unambiguous protein ID. Yet ESI-FTICR MS
instruments provide mass accuracy which is in some cases sufficient for protein identification using peptide mass information only (see articles about the
Accurate Mass Tags (AMT) approach from Richard Smith’s group).
(B)
The protein is a mammalian myoglobin, most probably - Equus burchelli (Plains zebra), with the Mascot score of 143 (significance threshold – 78) (see
the screenshots below). The unassigned peeks are sodium adducts, and see the link below for more information:
http://www.ionsource.com/tutorial/protID/fingerprint.htm
http://www.ionsource.com/tutorial/protID/fingerprintscreenshot.htm#Aldente%20Results%20Page