TY - JOUR
T1 - Normalization of relative peptide ratios derived from in-gel digests
T2 - Applications to protein variant analysis at the peptide level
AU - Bergen, H. Robert
AU - Muddiman, David C.
AU - O'Brien, John F.
AU - Hoyer, James D.
PY - 2005
Y1 - 2005
N2 - The ability to detect protein variants and post-translational modifications by mass spectrometry has become increasingly important. Unfortunately, the ability to detect variants in large intact proteins (>80 000 Da) is limited. Even in the analysis of smaller proteins, algorithms are required to determine the presence of a 2 Da mass shift in an intact 13 kDa protein because the isotopic distribution of the multiply charged ions of the variant overlaps the wild-type distribution. Fortunately, most modern instruments are capable of detecting variants in tryptic peptides derived from intact proteins. If a single common variant protein is known, the presence of a variant tryptic peptide can be easily demonstrated. A more difficult issue is the case where a multiplicity of peptides with multiple amino acid substitutions can be associated with pathology. In these cases a decrease in the relative amount of a variant peptide relative to other internal tryptic fragments would be diagnostic. However, the variability associated with the analysis of in-gel or solution digests of proteins, related to efficiencies in digestion, extraction and ionization, confounds variant analysis at the peptide level. A strategy was developed to normalize for this variability by utilizing multiple isotopically labeled internal standards for multiple peptides derived from the same protein. Erythrocyte spectrin from 36 normal and 25 abnormal osmotic fragility samples was analyzed as a test case. Three isotopically labeled target peptides comprising the α/β-spectrin self-association sites were added to purified digested α-spectrin. The utilization of multiple internal standards demonstrates the capability to normalize for sample variability due to ionization efficiency, solvent effects, digestion and extraction efficiency.
AB - The ability to detect protein variants and post-translational modifications by mass spectrometry has become increasingly important. Unfortunately, the ability to detect variants in large intact proteins (>80 000 Da) is limited. Even in the analysis of smaller proteins, algorithms are required to determine the presence of a 2 Da mass shift in an intact 13 kDa protein because the isotopic distribution of the multiply charged ions of the variant overlaps the wild-type distribution. Fortunately, most modern instruments are capable of detecting variants in tryptic peptides derived from intact proteins. If a single common variant protein is known, the presence of a variant tryptic peptide can be easily demonstrated. A more difficult issue is the case where a multiplicity of peptides with multiple amino acid substitutions can be associated with pathology. In these cases a decrease in the relative amount of a variant peptide relative to other internal tryptic fragments would be diagnostic. However, the variability associated with the analysis of in-gel or solution digests of proteins, related to efficiencies in digestion, extraction and ionization, confounds variant analysis at the peptide level. A strategy was developed to normalize for this variability by utilizing multiple isotopically labeled internal standards for multiple peptides derived from the same protein. Erythrocyte spectrin from 36 normal and 25 abnormal osmotic fragility samples was analyzed as a test case. Three isotopically labeled target peptides comprising the α/β-spectrin self-association sites were added to purified digested α-spectrin. The utilization of multiple internal standards demonstrates the capability to normalize for sample variability due to ionization efficiency, solvent effects, digestion and extraction efficiency.
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U2 - 10.1002/rcm.2134
DO - 10.1002/rcm.2134
M3 - Article
C2 - 16155979
AN - SCOPUS:25644458321
SN - 0951-4198
VL - 19
SP - 2871
EP - 2877
JO - Rapid Communications in Mass Spectrometry
JF - Rapid Communications in Mass Spectrometry
IS - 19
ER -