Reporting of Impurities, Contaminants, and Degradants

ProMass can automatically report the presence of impurities, contaminants, or degradants in deconvoluted spectra. Any component which is not a target mass is a putative impurity, contaminant, or degradant. Species including known cation or solvent adducts, and labile functional groups can be included in the automatic assignment. The automatically assigned components are listed in the Presumed Identity column of the Deconvolution Peak Report, which is available from the [Deconvolution Peak Report] hyperlink in the ProMass report for each sample. In order for ProMass to automatically assign known impurities, contaminants, or degradants, the following conditions must be met:

ProMass must be run in automated mode from the Xcalibur sample list.
At least one target mass must be specified in the sample list, either explicitly, or by including a BioSequence string and sequence type definition.
Masses for known impurities must be configured in the znova_masses.ini configuration file. Several common impurities and contaminants have already been defined for you, but it is simple to add new ones by editing this file with Notepad.
User residues, which are defined in the Target Info field of the sample list using the /mod/ = mass format, are automatically searched for as potential known components. The existence of plus and minus user-defined masses are considered.

ProMass will look for impurity masses for each target mass that is specified by searching the appropriate list in the znova_masses.ini file. Each impurity is identified by a descriptive string and a mass difference from the target mass. For example, consider the list of configured impurities below, which would be typical for oligonucleotide analysis:

[Impurities]
*Na adduct = 22
*K adduct = 38
*HFIPA adduct = 168
A depurination = -135
C depyrimidination = -111
G depurination = -151
T depyrimidination = -126
Minus Phos = -79.98
Minus A = -313.209
Minus C = -289.184
Minus G = -329.208
Minus T = -304.196
Plus DMF = 55

From the example above, a sodium adduct is identified as any mass peak that is +22 Da relative to a target mass. Depurination of an adenine base is identified as any peak producing a -135 Da decrease with respect to a target mass.

If the impurity definitition is preceded by a ' * ', the impurity is treated as an adduct and the abundance of that particular species will be ignored in the purity estimate calculations. The purity estimate for each target mass is reported on the top-level summary page and on the detailed report page for each sample. The purity estimate is a percentage based on the %total abundance of the target mass in the spectrum corrected for all adducts and normalized by peak area percent. For example, if a target mass with 50% total abundance has a Na adduct peak which is 10% abundance, and a chromatogram area percent of 20%, then the purity estimate = (50+10)*0.2 = 12%.

An example Deconvolution Peak Report is shown below. Note how the impurities are identified in the Presumed Identity column of the peak report.

Mass Peak List Sorted by Intensity:
Mass(Da)    +- Std. Dev.    Intensity    Score    Delta Mass   %Relative   %Total   Presumed Identity
  5494.3         0.3        2.45E+005    15.56         0.0       100.0      59.1     Target Mass: 5494.6
  5204.9         0.3        4.60E+004    10.00      -289.4        18.8      11.1     5494.6: Minus C
  4555.0         0.1        2.38E+004     4.67      -939.3         9.7       5.8     
  5181.1         0.4        2.17E+004     8.85      -313.2         8.9       5.2     5494.6: Minus A
  5358.6         0.3        1.93E+004     6.04      -135.7         7.9       4.7     5494.6: A depurination
  4602.5         0.3        1.26E+004     6.88      -891.8         5.1       3.0     
  6186.5         1.0        1.07E+004     5.63       692.2         4.4       2.6     
  5165.8         0.5        8.07E+003     6.05      -328.5         3.3       1.9     5494.6: Minus G
  5410.9         0.9        5.85E+003     2.87       -83.4         2.4       1.4     
  6473.0         1.0        5.42E+003     6.59       978.7         2.2       1.3     
  5528.5         0.8        5.38E+003     6.39        34.2         2.2       1.3     
  5343.7         0.4        3.85E+003     5.01      -150.6         1.6       0.9     5494.6: G depurination
  5547.3         0.9        3.65E+003     5.55        53.0         1.5       0.9     5494.6: Plus DMF
  5382.0         0.4        3.25E+003     4.79      -112.3         1.3       0.8     5494.6: C depyrimidination

Mass Peak List Sorted by Mass:
Mass(Da)    +- Std. Dev.    Intensity    Score    Delta Mass   %Relative   %Total   Presumed Identity
  4555.0         0.1        2.38E+004     4.67      -939.3         9.7       5.8     
  4602.5         0.3        1.26E+004     6.88      -891.8         5.1       3.0     
  5165.8         0.5        8.07E+003     6.05      -328.5         3.3       1.9     5494.6: Minus G
  5181.1         0.4        2.17E+004     8.85      -313.2         8.9       5.2     5494.6: Minus A
  5204.9         0.3        4.60E+004    10.00      -289.4        18.8      11.1     5494.6: Minus C
  5343.7         0.4        3.85E+003     5.01      -150.6         1.6       0.9     5494.6: G depurination
  5358.6         0.3        1.93E+004     6.04      -135.7         7.9       4.7     5494.6: A depurination
  5382.0         0.4        3.25E+003     4.79      -112.3         1.3       0.8     5494.6: C depyrimidination
  5410.9         0.9        5.85E+003     2.87       -83.4         2.4       1.4     
  5494.3         0.3        2.45E+005    15.56         0.0       100.0      59.1     Target Mass: 5494.6
  5528.5         0.8        5.38E+003     6.39        34.2         2.2       1.3     
  5547.3         0.9        3.65E+003     5.55        53.0         1.5       0.9     5494.6: Plus DMF
  6186.5         1.0        1.07E+004     5.63       692.2         4.4       2.6     
  6473.0         1.0        5.42E+003     6.59       978.7         2.2       1.3

Because impurities and contaminants are expected to be different for oligonucleotides vs. proteins, the impurity list is linked to the type of BioSequence which has been specified in the Xcalibur sample list. If you do not enter a BioSequence string and define the type of sequence, ProMass will use the list of generic impurities under the heading labeled [Impurities] in the znova_masses.ini file. For more information about editing this file see the ZNova Mass Configuration File help topic.