Identification of de novo synthesized and relatively older proteins: accelerated oxidative damage to de novo synthesized apolipoprotein A-1 in type 1 diabetes.
OBJECTIVE
The accumulation of old and damaged proteins likely contributes to complications of diabetes, but currently no methodology is available to measure the relative age of a specific protein alongside assessment of posttranslational modifications (PTM). To accomplish our goal of studying the impact of insulin deficiency and hyperglycemia in type 1 diabetes upon accumulation of old damaged isoforms of plasma apolipoprotein A-1 (ApoA-1), we sought to develop a novel methodology, which is reported here and can also be applied to other specific proteins.


METHODS
To label newly synthesized proteins, [ring-(13)C(6)]phenylalanine was intravenously infused for 8 h in type 1 diabetic participants (n = 7) during both insulin treatment and 8 h of insulin deprivation and in nondiabetic participants (n = 7). ApoA-1 isoforms were purified by two-dimensional gel electrophoresis (2DGE) and assessment of protein identity, PTM, and [ring-(13)C(6)]phenylalanine isotopic enrichment (IE) was performed by tandem mass spectrometry.


RESULTS
Five isoforms of plasma ApoA-1 were identified by 2DGE including ApoA-1 precursor (pro-ApoA-1) that contained the relatively highest IE, whereas the older forms contained higher degrees of damage (carbonylation, deamidation) and far less IE. In type 1 diabetes, the relative ratio of IE of [ring-(13)C(6)]phenylalanine in an older isoform versus pro-ApoA-1 was higher during insulin deprivation, indicating that de novo synthesized pro-ApoA-1 more rapidly accumulated damage, converting to mature ApoA-1.


CONCLUSIONS
We developed a mass spectrometry-based methodology to identify the relative age of protein isoforms. The results demonstrated accelerated oxidative damage to plasma ApoA-1, thus offering a potential mechanism underlying the impact of poor glycemic control in type 1 diabetic patients that affects a patient's risk for vascular disease.
Jaleel
Abdul
A
Division of Endocrinology, Mayo Clinic, Rochester, Minnesota, USA.
Henderson
Gregory C
GC
Madden
Benjamin J
BJ
Klaus
Katherine A
KA
Morse
Dawn M
DM
Gopala
Srinivas
S
Nair
K Sreekumaran
KS
D000328:Adult; D000595:Amino Acid Sequence; D016632:Apolipoprotein A-I / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015992:Body Mass Index; D003922:Diabetes Mellitus, Type 1 / Q000097:blood*; D003924:Diabetes Mellitus, Type 2 / Q000097:blood; D006442:Glycated Hemoglobin / Q000378:metabolism; D006801:Humans; D007004:Hypoglycemic Agents / Q000627:therapeutic use; D007328:Insulin / Q000097:blood / Q000627:therapeutic use; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000097:blood / Q000737:chemistry / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012016:Reference Values
D016632:Apolipoprotein A-I; D006442:Glycated Hemoglobin; D007004:Hypoglycemic Agents; D007328:Insulin; D020033:Protein Isoforms