Version: 1.0
“R”-program that corrects 13C mass isotopomers spectra of metabolites for natural occurring isotopes and peaks overlapping
MIDcor is an “R”-program that performs a primary analysis of isotopic isomers (isotopomers) distribution obtained by Gas Cromatography coupled with Mass Spectrometry (GCMS). The aim of this analysis is to have a correct distribution of isotopes originated from substrates that are artificially enriched with specific isotopes (usually 13C). To this end the program performs a correction for natural occurring isotopes and also correction for “impurities” of the assay media that give peaks overlapping with the spectra of analyzed labeled metabolites. This program offers two ways of corrections of “impurities” resulted from overlapping the assayed mass isotopomer distribution with peaks produced either by unknown metabolites in the media, or by different fragments produced by the assayed metabolite.
This program offers two ways of corrections of “impurities” resulted from overlapping the assayed mass isotopomer distribution with peaks produced either by unknown metabolites in the media, or by different fragments produced by the assayed metabolite.
- primary processing of 13C mass isotopomer data obtained with GCMS
- Preprocessing
- Statistical Analysis
- Workflows
- Isotopic Labelling Analysis / 13C
- MS
- correction for H+ loss produced by electron impact, natural occurring isotopes, peaks overlapping that depends on isotopic composition of metabolites
- Vitaly Selivanov (Universitat de Barcelona)
- N/A
- As independent program. MIDcor itself does not require installation. Standing in the MIDcor directory enter in R environment with the command:
''' R '''
read the necessary functions:
''' source("lib.R")
source("midcor.R")'''
Analysis the raw mass spectra presented in the file "filename" using the functions available in MIDcor:
correct("filename",samb=1,samf=3,cndb=4,cndf=9)
Here "filename" is the name of the input file contaiming raw mass spectra. The parameters samb,samf,cndb,cndf are the positions of the initial and final characters in the row names designating the biological sample and conditions correspondingly Here is an example of an input file. Comments are included between * *, they should not be present in a real input file.
carbons_total 18 * Total number of carbons in the derivated fragment *
fragment 4 <number of carbons in the assayed metabolite (or its fragment) >
silicium 3
sulfur 0
m/z 417 418 419 420 421 422 423
commer_01.D 911 144256 52288 24832 9517 2647 1037
commer_02.D 929 127872 46416 21664 7421 2285 688
commer_03.D 1039 135296 49192 22760 7967 2202 681
natural_01.D 782 109024 39368 18592 7084 1894 674
natural_02.D 1059 134528 49200 22968 7643 2027 644
natural_03.D 1177 151808 54456 25152 8434 2396 773
B10n8_01.D 867 105080 40672 29472 22152 68248 21936
B10_02.D 1294 148416 56784 41776 29216 95936 31608
B10_03.D 1311 150528 57464 42104 28776 95184 30776
B11n8_01.D 673 67576 25768 19064 16002 44320 14340
B11_02.D 1113 108120 41208 30208 25192 71648 23056
B11_03.D 1056 98024 37736 27880 22280 65240 21328
B12n8_01.D 1091 250304 95240 74224 48360 180288 57240
B12_02.D 918 117448 45728 36096 28784 95920 30728
B12_03.D 1540 194560 75856 58952 44088 152704 48896
B4_c801.D 1296 239744 87064 43832 16085 4421 1499
B4_02.D 1468 283968 103032 52080 16744 4777 1367
B4_03.D 1598 300224 108576 54832 17880 5120 1375
B5_c801.D 1277 212160 78016 38984 13210 3783 1105
B5_02.D 1895 331392 120048 60376 17800 5044 1326
B5_03.D 1835 297664 108208 55768 17232 4738 1194
B6_c801.D 989 146240 53128 26744 12221 3627 1254
B6_02.D 516 23536 8442 4540 5035 1705 809
B6_03.D 515 2997 1204 719 4041 1435 657
R10n8_01.D 856 110320 43184 34032 22680 66200 21704
R10_02.D 1197 163072 65632 50376 31136 98704 31080
R10_03.D 1421 175552 69144 53384 32440 103688 32432
R11n8_01.D 790 108040 42136 32840 21728 63776 20312
R11_02.D 1214 169984 67200 52344 33120 100352 32424
R11_03.D 1207 161856 63624 49032 30968 96616 30776
R12n8_01.D 791 111560 44456 33976 21560 64456 20120
R12_02.D 957 141440 56584 43480 29032 84056 26656
R12_03.D 1077 154112 60488 47360 31152 92824 29616
R4_c801.D 242 189 293 169 150 92 87
R4_02.D 446 255 302 176 162 88 85
First four lines provide information necessary for the correction for natural occurring isotopes. The fifth line indicates the m/z range for the spectra starting from M-1 isotope. The next lines consist of a word representing a name of a given sample and the intensities of peaks corresponding to the indicated m/z values. The last line is recognized by “fin” in the beginning.
First of all, MIDcor corrects the spectra for proton lost due to electronic impact, defined by the ratio (M-1)/M. Then it normalizes the intensities in corrects each row and corrects for the natural occurrence of isotopes. It writes the results in "filename_c" under the title "*** MID for each injection, corrected only for natural 13C, 29,30Si, 33,34S ***"
Then MIDcor sums the corresponding intensities presented in multiple injections from the same biological samples (technical replicates). In the example provided it recognizes an individual biological sample by three first characters. The same first three characters indicate that the corresponding injections are from the same sample. Respectively, the parameters samb and samf should have values 1 and 3. In principle, samples can be characterized by characters in different positions, and, respectively the parameters samb and samf can have different values. After summing the technical replicates, it corrects the sums for natural occurring isotopes and writes the result under the title "*** Summed injections for each plate, corrected only for natural 13C, 29,30Si, 33,34S **".
After summation, the technical replicates the set of spectra contains only different biological replicates. Then MIDcor corrects them for peaks overlapping and finds the mean values and standard deviations among the replicates referring to the same conditions of incubation. The program recognizes the same conditions by the same characters that indicate the conditions. In the example provided it is the next two characters. In this case, the parameters cndb and cndf should have values 4 and 5 correspondingly. It shows the results under the titles: *** Statistics, samples fully corrected **
To correct the peaks overlapping, MIDcor calculates the difference D between the measured mass spectra and the ones theoretically for the unlabeled samples. The unlabeled samples are recognized by the substring "commer" (for commercial preparation in minimal media containing only the substance and chemicals for its derivatization) or "natural" (containing the unlabeled substance in full media of cell incubation before the incubation starts).
Midcor performs the correction for peaks overlapping in labeled samples by one of two ways; either (i) subtract the obtained D without any change from the normalized measured spectra, or (ii) change the obtained D before its subtraction. The change includes shift D by the number of 13C labels obtained from artificially 13C enriched substrates and taking the part of D equal to the portion of corresponding mass isotopomer. To decide which of the two ways should be taken MIDcor compares the two D-vectors obtained for "commercial" or "natural" unlabeled samples. If the max value of D obtained for "natural" media is more than 0.5% larger than the one obtained in minimal media than MIDcor chooses the way (i) with D obtained for the "natural" conditions. In opposite cases, it chooses the way (ii).
Finally, it writes the D obtained for the "natural" conditions in the output file under the title
*** Correction factor: **
If a user decides to check whether some "isotopic effect" takes place, he/she should provide the set of measured intensities for an "artificially" labeled sample with labeling pattern a priori known (it could be a commercial preparation). The name of such a sample should contain the substring "fitf". Midcor fits the known 13C mass isotopomer pattern thus determining the factor of "isotopic effect"
- “MIDcor”, an R-program for deciphering mass interferences in mass spectra of metabolites enriched in stable isotopes. Submitted to BMC bioinformatics.