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Transcript of The Student Research and Scholarship Center Grove School of Engineering, And Pathways Bioinformatics...
The Student Research and Scholarship CenterGrove School of Engineering,
And Pathways Bioinformatics Center , CCNYPresent
Winter Bioinformatics Workshop 10am-6pm, January 20 – 21, 2009
Location: Marshak, Room MR-044
Contact info: [email protected], 212-650-8870
Invited Speaker
Cristina C. Clement, Ph.D. Albert Einstein College of Medicine, Pathology Department
Topic: Bioinformatics tools for proteins identification from primary sequence databases
using mass spectrometry data
January 21, Marshak Building, Room MR-044
1:00pm-2:00pm: Presentation 2:30pm-4:00pm: Online practice
Accuracy & Resolution in Mass SpectroscopyWhen low molecular weight samples are being analysed using relatively low resolution mass spectrometers, it is common to work with "nominal" mass values, calculated from integer atomic weights. That is, H=1, C=12, N=14, O=16, etc. Nominal mass is rarely used in peptide and protein work because the cumulative error of approximating atomic weights with integers becomes unacceptable.
The presence of isotopes at their natural abundances makes it essential to define whether an experimental mass value is an "average" value, equivalent to taking the centroid of the complete isotopic envelope, or a "monoisotopic" value, the mass of the first peak of the isotope distribution.
For peptides and proteins, the difference between an average and a monoisotopic weight is approximately 0.06%. This is a significant difference when even the most modest instruments are capable of measuring the mass of a small peptide with an accuracy of a fraction of a Dalton. For example, peptide HLKTEAEMK has an average molecular weight of 1086.28 and a monoisotopic weight of 1085.55. At a mass resolution of 5000, the isotopic envelope has this appearance:
m/z
Mass resolution is the dimensionless ratio of the mass of the peak divided by its width. Usually, the peak width is taken as the full width at half maximum intensity, (fwhm). However, this definition of peak width is only a convention, and you may also encounter data acquired on magnetic sector instruments where the resolution has been calculated using the peak width at 5% maximum intensity.
To measure a monoisotopic molecular weight requires (i) sufficient mass resolution to resolve the the isotopic distribution (ii) sufficient signal to noise to be able to identify the first peak of the envelope with confidence. For a small peptide, the first peak (often referred to as the 12C peak) is also the most intense peak. This is not the case for larger molecules. The following two examples show the isotopic envelopes for a small protein (insulin) and a larger protein (BSA):
It would be extremely difficult to measure a monoisotopic mass for BSA, and it is far from routine to measure a monoisotopic mass for insulin. In practice, most instruments report monoisotopic molecular weights up to a certain cut-off point. Above this
cut-off, isotopic envelopes are centroided as a whole to provide average mass values.
Accuracy & Resolution in Mass Spectroscopy
m/z m/z
Accuracy & Resolution in Mass Spectroscopy
The factor which complicates any general discussion of resolution optimisation is that some types of mass analyser have a trade-off between resolution and sensitivity, while others do not.
Where a monoisotopic peak for a single molecular species can be resolved, mass accuracy tends to follow resolution. This is because the narrower the peak, the less the significance of errors due to variations in the peak shape. So, if unit mass resolution is possible, then the more resolution the better ... unless there is a sensitivity trade-off.
If unit mass resolution is not possible, then there is little benefit to exceeding the instrument resolution at which the isotopic envelope can be defined without significant broadening. For example, the following figure shows the molecular ion of glucagon at resolutions of 1000 (blue), 3000 (red), 10,000 (green) and 30,000 (black).
For an average mass measurement, and where there is no trade-off between sensitivity and resolution, the accuracy at 3000 resolution (red) will be just as good as at higher resolution. On an instrument where a trade-off exists, using a resolution greater than 3000 is very likely to degrade mass accuracy.
m/z
In this MS-MS approach (depicted above) a first mass spectrometer (MS-1) that employs a quadrupole mass filter is tuned to allow only the analyte ion of interest (e.g. red above) through.
This is then taken into a collision cell where Argon is used to fragment the analyte, and the so-called daughter ions are then swept into a second time-of-flight MS (MS-2) where they are separated and detected.
The most common peptide fragments observed in low energy collisions are a, b and y ions, as described in the figure above. The b ions appear to extend from the amino terminus, sometimes called the N-terminus, and y ions appear to extend from the carboxyl terminus, or C-terminus. While readily observed and diagnostic for b ions, a ions occur at a lower frequency and abundance in relation to b ions. The a ions are often used as a diagnostic for b ions, such that a-b pairs are often observed in fragment spectra. The a-b pairs are separated by 28u, the mass for the carbonyl, C=O. The fragment types listed above are the most common fragments observed with ion trap, triple quadrupole, and q-TOF mass spectrometers.
De novo sequencing of peptides
This is an MS/MS spectrum of the tryptic peptide GLSDGEWQQVLNVWGK. This data was collected on an ion trap mass spectrometer. This spectrum will be the subject of our first unblinded de novo sequencing example.
The sequence of the peptide is determined by the mass difference between these peaks.
The b fragment peaks are labeled from the amino to the carboxyl terminus. The fragment containing only the amino terminal amino acid is termed b1. The fragment containing the first two amino terminal amino acids is termed the b2 ion, and so forth. The nomenclature is very simple to follow.
You can calculate the mass of any b ion, basically it is the mass of the shortened peptide (M)-17 (OH) = b ion m/z or more simply M-17 = b ion m/z.
Shows the first six b ions in a little bit more detail. The b ion m/z value is basically the mass of the peptide minus OH, or -17u.
Loss of Ammonia and Water
y and b ion fragments containing the amino acid residues R, K, Q, and N may appear to lose ammonia, -17.-
y and b ion fragments containing the amino acid residues S, T, and E may appear to lose water, -18. In the case of glutamic acid, E must be at the N-terminus of the fragment for this observation to be made.
Spectral Intensity Rules
b ion intensity will drop when the next residue is P, G or also H, K, and R.-
Internal cleavages can occur at P and H residues. An internal cleavage fragment is a fragment that appears to be a shortened peptide with P and or H at its amino terminus, for example the peptide EFGLPGLQNK may display the b ions PGLQNK, PGLQN, PGLQ, etc. These are the result of a double cleavage event. The y ion intensity will often be the most
prominent peak in the spectrum. -
It is common for b and y ions or y and b ions to swap intensity when a P is encountered in a sequence. This can also be true when the basic residues H, K, or R are encountered in the sequence.
- When a cleavage appears before or after R, the -17 (loss of ammonia) peak can be more prominent than the
corresponding y or b ion.-
When encountering aspartic acid in a sequence, the ion series can die out.
Amino Acid Composition
It is possible to observe immonium ions at the low end of the spectrum that can give a clue to the amino acid composition of a peptide. One caveat is that if you do not see an immonium ion for a particular amino acid, this does
not mean that that amino acid is absent from the sequence.
Isobaric MassLeucine and Isolucine have isobaric masses and cannot be differentiated in a low energy collision.
When we see this mass difference in a spectrum we will label it X or Lxx, adopting the Hunt nomenclature.
- Lysine and Glutamine have near isobaric masses, 128.09496 and 128.05858 respectively. The
delta mass is 0.03638 this difference can be used to differentiate K from Q on a mass spectroneter capable of higher mass accuracy and resolution, such as a q-TOF mass spectrometer. Usually triple quadrupole or ion trap mass spectrometers are incapable of this feat. On a lower mass
accuracy mass spectrometers an acetylation can be performed to shift the mass of lysine by 42u. If you like to live dangerously, and we do not, one can assume that a 128 mass shift internally on a
tryptic peptide is a glutamine unless followed by a proline or sometimes aspartic acid. Other instances of internal lysines left standing after a tryptic digest (this is our personal observation) is
when double lysines occur in a sequence, so be careful.-
There are instances where two residues will nearly equal the mass of a single residue, or a modified residue will nearly equal the mass of another amino acid.
More Rules
When starting a de novo sequencing project, start at the high mass end of the spectrum; the lower number of peaks at this end often makes it easier to start sequencing.
- The region 60 u below the parent mass can be confounded by multiple water and ammonia losses,
be careful. Realize that glycine may be your first amino acid and may fall in this region. -
Do you want to know if your tryptic peptide ends in a K or an R? Look for the diagnostic y1 ions at the low end of the spectrum, you may observe 147 for K or 175 for R.
- The b1 fragment is seldom observed making it difficult to determine the order of the first two N-terminal amino acids in a peptide sequence. Solutions for this problem can include a one step
Edman degradation or an acetylation.-
Once you know the mass of a b or y ion the corresponding y or b ion can be calculated using the following formulas.
- y = (M+H)1+ - b +1
- b = (M+H)1+ - y +1
Mascot Search OverviewMascot is a powerful search engine which uses mass spectrometry data to identify proteins from primary sequence databases. While a number of similar programs available, Mascot is unique in that it integrates all of the proven methods of searching. These different search methods can be categorised as follows:
Peptide Mass Fingerprint in which the only experimental data are peptide mass values.Sequence Query in which peptide mass data are combined with amino acid sequence and composition information. A super-set of a sequence tag query.MS/MS Ion Search using uninterpreted MS/MS data from one or more peptides.
The general approach for all types of search is to take a small sample of the protein of interest and digest it with a proteolytic enzyme, such as trypsin. The resulting digest mixture is analysed by mass spectrometry.
Different types of mass spectrometer have different capabilities. A simple instrument will measure a set of molecular weights for the intact mixture of peptides. An instrument with MS/MS capability can additionally provide structural information by recording the fragment ion spectrum of a peptide. Usually, the digest mixture will be separated by chromatography prior to MS/MS analysis, so that MS/MS spectra from individual peptides can be measured.The experimental mass values are then compared with calculated peptide mass or fragment ion mass values, obtained by applying cleavage rules to the entries in a comprehensive primary sequence database. By using an appropriate scoring algorithm, the closest match or matches can be identified. If the "unknown" protein is present in the sequence database, then the aim is to pull out that precise entry. If the sequence database does not contain the unknown protein, then the aim is to pull out those entries which exhibit the closest homology, often equivalent proteins from related species.The sequence databases that can be searched on this server are:MSDB is a comprehensive, non-identical protein sequence database maintained by the Proteomics Department at the Hammersmith Campus of Imperial College London. MSDB is designed specifically for mass spectrometry applications. NCBInr is a comprehensive, non-identical protein database maintained by NCBI for use with their search tools BLAST and Entrez. The entries have been compiled from GenBank CDS translations, PIR, SWISS-PROT, PRF, and PDB. SwissProt is a high quality, curated protein database. Sequences are non-redundant, rather than non-identical, so you may get fewer matches for an MS/MS search than you would from a comprehensive database, such as MSDB or NCBInr. SwissProt is ideal for peptide mass fingerprint searches. dbEST is the division of GenBank that contains "single-pass" cDNA sequences, or Expressed Sequence Tags, from a number of organisms. During a Mascot search, the nucleic acid sequences are translated in all six reading frames. dbEST is a very large database, and is divided into three sections: EST_human, EST_mouse, and EST_others. Even so, searches of these databases take far longer than a search of one of the non-redundant protein databases. You should only search an EST database if a search of a protein database has failed to find a match.
http://www.matrixscience.com/
All matches to this query
ScoreMr(calc): Delta Sequence
46.1 1604.8535 0.0046 THRIHWESASLLR
31.0 1605.7594 -0.9012 SSNSSPSRXSLGQLSE
30.2 1604.8133 0.0448 PRFCASLAGGAWLSGL
28.8 1605.7006 -0.8424 ETKEGTEGEGLQEEA
28.8 1605.7006 -0.8424 ETKEGTEGEGLQEEX
28.8 1604.7722 0.0859 GGSLYEAPVSYTFSK
28.5 1605.8627 -1.0045 APVIPAPWEAKAGGSR
27.6 1604.7715 0.0866 EEGTAASLADIMEIR
27.0 1603.8318 1.0264 HVLAHSESINVIAQS
26.9 1604.7464 0.1118 SSSILAMRDEQSNPA
Protein ViewMatch to: gi|226159 Score: 46: complement C3fNominal mass (Mr): 2020; NCBI BLAST search of gi|226159 against nrUnformatted sequence string for pasting into other applicationsTaxonomy: Homo sapiensNo enzyme cleavage specificitySequence Coverage: 76%Matched peptides shown in Bold Red 1 SSKITHRIHW ESASLLR
Peptide ViewMS/MS Fragmentation of THRIHWESASLLRFound in gi|226159, complement C3fMatch to Query 1: 1604.858172 from(535.960000,3+)
Mascot search of MS/MS databasesCristina C. Clement, unpublished results
Monoisotopic mass of neutral peptide Mr(calc): 1604.8535Ions Score: 46 Expect: 5.4 Matches (Bold Red): 43/134 fragment ions using 51 most intense peaks
# b b++ b* b*++ b0 b0++ Seq.
y y++ y* y*++ y0 y0++ #
1 102.0550 51.5311 84.0444 42.5258 T 13
2 239.1139 120.0606 221.1033111.0553
H1504.8132
752.9102
1487.7866
744.3969
1486.8026
743.9049
12
3 395.2150 198.1111378.1884
189.5979
377.2044189.1058
R1367.7542
684.3808
1350.7277
675.8675
1349.7437
675.3755
11
4 508.2990 254.6532491.2725
246.1399
490.2885245.6479
I1211.6531
606.3302
1194.6266
597.8169
1193.6426
597.3249
10
5 645.3580 323.1826628.3314
314.6693
627.3474314.1773
H1098.5691
549.7882
1081.5425
541.2749
1080.5585
540.7829
9
6 831.4373 416.2223814.4107
407.7090
813.4267407.2170
W 961.5102481.2587
944.4836472.7454
943.4996472.2534
8
7 960.4799 480.7436943.4533
472.2303
942.4693471.7383
E 775.4308388.2191
758.4043379.7058
757.4203379.2138
7
8 1047.5119 524.25961030.4853
515.7463
1029.5013
515.2543
S 646.3882323.6978
629.3617315.1845
628.3777314.6925
6
9 1118.5490 559.77811101.5225
551.2649
1100.5384
550.7729
A 559.3562280.1817
542.3297271.6685
541.3457271.1765
5
10
1205.5810 603.29421188.5545
594.7809
1187.5705
594.2889
S 488.3191244.6632
471.2926236.1499
470.3085235.6579
4
11
1318.6651 659.83621301.6385
651.3229
1300.6545
650.8309
L 401.2871201.1472
384.2605192.6339
3
12
1431.7492 716.37821414.7226
707.8649
1413.7386
707.3729
L 288.2030144.6051
271.1765136.0919
2
13
R 175.1190 88.0631 158.0924 79.5498 1
Cristina C. Clement, unpublished results
LII_3 #8410 RT: 48.42 AV: 1 NL: 1.95E5T: ITMS + p NSI d Z ms [ 546.00-556.00]
546 547 548 549 550 551 552 553 554 555 556m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
549.80
550.24
550.72
551.24555.26551.82546.70 548.88547.18 553.54548.40 552.64 555.74554.12
550.52-LTQ (2+)
LII_3 #8411 RT: 48.42 AV: 1 NL: 1.93E5T: ITMS + c NSI d Full ms2 [email protected] [ 140.00-1115.00]
200 300 400 500 600 700 800 900 1000 1100m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
775.48
324.25
961.56
646.52
540.90296.28
435.30
472.80757.55 924.45195.21 279.26 811.45401.51 611.35 698.42 971.52896.33 1054.20
Cristina C. Clement, unpublished results
All matches to this query
ScoreMr(calc): Delta Sequence
45.2 1097.5618 0.0237 HWESASLLR
30.0 1097.5651 0.0203 HGKEMDLLR
29.2 1098.4441 -0.8586 GFTFSASDMH
27.6 1097.5869 -0.0015 AFSFSSALIR
26.9 1098.4652 -0.8797 HSTYSSLMSS
26.7 1097.4989 0.0865 HGEEASSAIPT
24.6 1097.6094 -0.0240 HQGKLVFNR
24.1 1098.4764 -0.8910 HGEEGMGQGVV
24.0 1097.5353 0.0502 HGEKEEELK
24.0 1099.4274 -1.8420 QSQKSSMDSC
Protein ViewMatch to: gi|226159 Score: 42: complement C3fNominal mass (Mr): 2020; NCBI BLAST search of gi|226159 against nrUnformatted sequence string for pasting into other applicationsTaxonomy: Homo sapiens
No enzyme cleavage specificitySequence Coverage: 52%
Matched peptides shown in Bold Red
1 SSKITHRIHW ESASLLR
Peptide ViewMS/MS Fragmentation of HWESASLLRFound in gi|226159, complement C3fMatch to Query 1: 1097.585448 from(549.800000,2+)
Cristina C. Clement, unpublished results
Monoisotopic mass of neutral peptide Mr(calc): 1097.5618Ions Score: 45 Expect: 6.5 Matches (Bold Red): 26/70 fragment ions using 37 most intense peaks
# b b++ b0 b0++ Seq. y y++ y* y*++ y0 y0++ #
1138.066
269.5367 H 9
2324.145
5162.576
4 W
961.5102
481.2587
944.4836
472.7454
943.4996
472.2534
8
3453.188
1227.097
7435.177
5218.092
4E
775.4308
388.2191
758.4043
379.7058
757.4203
379.2138
7
4540.220
1270.613
7522.209
6261.608
4S
646.3882
323.6978
629.3617
315.1845
628.3777
314.6925
6
5611.257
2306.132
3593.246
7297.127
0A
559.3562
280.1817
542.3297
271.6685
541.3457
271.1765
5
6698.289
3349.648
3680.278
7340.643
0S
488.3191
244.6632
471.2926
236.1499
470.3085
235.6579
4
7811.373
3406.190
3793.362
8397.185
0L
401.2871
201.1472
384.2605
192.6339
3
8924.457
4462.732
3906.446
8453.727
1L
288.2030
144.6051
271.1765
136.0919
2
9 R175.119
088.0631
158.0924
79.5498 1
1 SSKITHRIHW ESASLLR - 1st hit for complement c3f
1 SSKITHRIHW ESASLLR - 2nd hit for complement c3f
Cristina C. Clement, unpublished results
Monoisotopic:733.32, Charge= +2
LII_3 #12446 RT: 72.23 AV: 1 NL: 7.50E3T: ITMS + p NSI d Z ms [ 729.00-739.00]
729 730 731 732 733 734 735 736 737 738 739m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rela
tive A
bundance
733.32
733.76
734.22
734.72
735.18729.38 731.20 737.22730.04 731.82 735.48 738.30
LII_3 #12447 RT: 72.23 AV: 1 NL: 6.88E2T: ITMS + c NSI d Full ms2 [email protected] [ 190.00-1480.00]
200 400 600 800 1000 1200 1400m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
645.41
445.28 574.35
758.45
1077.53821.45
1021.39905.49
1291.42561.34 1135.51388.31
331.27 1192.391360.78
Cristina C. Clement, unpublished results
Probability Based Mowse ScoreIons score is -10*Log(P), where P is the probability that the observed match is a random event.Individual ions scores > 56 indicate peptides with significant homology.Individual ions scores > 73 indicate identity or extensive homology (p<0.05).Protein scores are derived from ions scores as a non-probabilistic basis for ranking protein hits.
Peptide View MS/MS Fragmentation of DSGEGDFLAEGGGVRFound in gi|229185, fibrinopeptide AMatch to Query 1: 1464.625448 from(733.320000,2+)Data file C:\Documents and Settings\Laura Santambrogio\Desktop\rksah062708\LTQ\LTQ-ccc\LII_3RAW071608\12446Raw_2+.txt
# b b++ b0 b0++ Seq. y y++ y* y*++ y0 y0++ #1 116.0342 58.5207 98.0237 49.5155 D 15
2 203.0662 102.0368 185.0557 93.0315 S 1350.6284 675.8179 1333.6019 667.3046 1332.6179 666.8126 14
3 260.0877 130.5475 242.0771 121.5422 G 1263.5964 632.3018 1246.5699 623.7886 1245.5858 623.2966 13
4 389.1303 195.0688 371.1197 186.0635 E 1206.5749 603.7911 1189.5484 595.2778 1188.5644 594.7858 12
5 446.1518 223.5795 428.1412 214.5742 G 1077.5323 539.2698 1060.5058 530.7565 1059.5218 530.2645 11
6 561.1787 281.0930 543.1681 272.0877 D 1020.5109 510.7591 1003.4843 502.2458 1002.5003 501.7538 10
7 708.2471 354.6272 690.2366 345.6219 F 905.4839 453.2456 888.4574 444.7323 887.4734 444.2403 9
8 821.3312 411.1692 803.3206 402.1639 L 758.4155 379.7114 741.3890 371.1981 740.4050 370.7061 8
9 892.3683 446.6878 874.3577 437.6825 A 645.3315 323.1694 628.3049 314.6561 627.3209 314.1641 7
10 1021.4109 511.2091 1003.4003 502.2038 E 574.2944 287.6508 557.2678 279.1375 556.2838 278.6455 6
11 1078.4324 539.7198 1060.4218 530.7145 G 445.2518 223.1295 428.2252 214.6162 5
12 1135.4538 568.2305 1117.4433 559.2253 G 388.2303 194.6188 371.2037 186.1055 4
13 1192.4753 596.7413 1174.4647 587.7360 G 331.2088 166.1081 314.1823 157.5948 3
14 1291.5437 646.2755 1273.5331 637.2702 V 274.1874 137.5973 257.1608 129.0840 2
15 R 175.1190 88.0631 158.0924 79.5498 1
Monoisotopic mass of neutral peptide Mr(calc): 1464.6481Ions Score: 123 Expect: 5.3e-07 Matches (Bold Red): 38/130 fragment ions using 34 most intense peaks
Cristina C. Clement, unpublished results
Score Mr(calc): Delta Sequence
123.5 1464.6481 -0.0227 DSGEGDFLAEGGGVR
79.8 1463.6641 0.9614 DSGEGDFLAEGGGVR
37.2 1464.6079 0.0176 LDLCQDSFPGNPTG
36.6 1467.6776 -3.0521 EMYRNLAQGRNV
36.6 1467.6776 -3.0521 EMYRNLAQGRNV
35.6 1464.6721 -0.0466 CARGWAFDIWGQG
33.7 1464.7606 -0.1352 SSVGTEMIITKAGR
33.5 1465.6645 -1.0390 RSSGGETETTGQSAV
33.5 1466.6485 -2.0231 RSSGGETETTGQSAV
32.8 1464.6568 -0.0314 CARDQAFDIWGQG
1 MFSMRIVCLV LSVVGTAWTA DSGEGDFLAE GGGVRGPRVV ERHQSACKDS 51 DWPFCSDEDW NYKCPSGCRM KGLIDEVNQD FTNRINKLKN SLFEYQKNNK 101 DSHSLTTNIM EILRGDFSSA NNRDNTYNRV SEDLRSRIEV LKRKVIEKVQ 151 HIQLLQKNVR AQLVDMKRLE VDIDIKIRSC RGSCSRALAR EVDLKDYEDQ 201 QKQLEQVIAK DLLPSRDRQH LPLHSSLGDR ARLHLKTNKT AKKKKKKKKK
Cristina C. Clement, unpublished results
Bioinformatics Workshop project
Leading project supervisor: Cristina C. Clement, Ph.D.
Set up the MS/MS Ion search parameters using Mascot algorithmhttp://www.matrixscience.com/
Import txt. files of ms/ms data at the Mascot server ON LINE(3 independent txt files are provided/student)
01/21/2009
Analyze Mascot results
-Analyze Peptide View, understand the scoring functions -Analyze the Protein View, understand the sequence coverage
Re-analyze the txt MS/MS files using independent searching algorithmsat PROSPECT and PROFOUND webservers