The main focus of the serum amyloid A (SAA) family has been on the acute phase isoforms. However, the constitutive isoform (SAA4) may have a strong effect on the metabolism of human serum lipoproteins. In this study, the SAA4 protein was examined in the high-density lipoprotein fraction of both healthy and diseased individuals. Novel isoforms of SAA4 were detected using ultracentrifugation combined with solid-phase extraction and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Three truncated isoforms were identified as well as two glycosylated isoforms. Patterns of isoform distribution may be significant for assessment of cardiovascular risk as well as direction of patient treatment.

CONTEXT:Low birth weight is associated with increased cardiovascular disease in adulthood, and differences in the molecular weight, composition, and quantity of lipoprotein subclasses are associated with coronary artery disease.OBJECTIVE:To determine if there are novel patterns of lipoprotein heterogeneity in low-birth-weight infants.DESIGN, SETTING, AND PARTICIPANTS:Prospective study at a US medical center of a representative sample of infants (n = 163; 70 white and 93 black) born at 28 or more weeks of gestational age between January 3, 2000, and September 27, 2000. This sample constituted 20% of all infants born during the study period at this site.MAIN OUTCOME MEASURES:Plasma levels and particle sizes of lipoprotein subclasses and plasma concentrations of lipids, lipoproteins (high-density lipoprotein [HDL] and low-density lipoprotein [LDL]), and apolipoproteins.RESULTS:An elevated lipoprotein peak of a particle with density between 1.062 and 1.072 g/mL was identified using physical-chemical methods. This subclass of large HDL was enriched in apolipoprotein C-I (apo C-I). Based on the amount of the apo C-I-enriched HDL peak, 156 infants were assigned to 1 of 4 groups: 0 (none detected), 17%; 1 (possibly present), 41%; 2 (probably present), 22%; 3 (elevated), 19%. Infants in group 3, compared with those in the other 3 groups, had significantly (P<.001) lower mean birth weight (2683.7 vs 3307.1 g) and younger mean gestational age (36.2 vs 39.3 wk). After correction for age, infants in group 3 had significantly higher levels of total and large HDL cholesterol and of total and large LDL cholesterol and LDL particle number. However, infants in group 3 had lower levels of small HDL, very low-density lipoproteins, and triglycerides than infants in the other 3 groups. This lipoprotein profile differed from that in infants born small for gestational age, who had significantly higher triglyceride (P<.001) and apo B (P = .04) levels, but lower levels of total and large HDL cholesterol (P<.001) and apo A-I (P<.001).CONCLUSIONS:Because apo C-I-enriched HDL, and purified apo C-I alone, promotes apoptosis in vitro, increased amounts of this particle may have physiological significance and identify a novel group of low-birth-weight infants apparently distinct from traditionally classified small-for-gestational-age infants.

The proteins associated with the circulating lipoproteins in the blood function not only for mediating lipid metabolism but also for maintaining structural stability of the micellelike structure. Any modifications of these proteins, by mutation or posttranslational modification, could compromise the function of these proteins and contribute to the development of cardiovascular disease. Because of the presence of extensive lipophilic domains, these proteins, when recovered from the lipoprotein particle (apolipoproteins) present an analytical challenge because of low solubility and proclivity toward aggregate formation. Our goal is to characterize these proteins by a combination of high-accuracy pI measurement coupled with MALDI analysis. In this report, we demonstrate the high resolution of immobilized pH gradient isoelectric focusing (IPG-IEF) for the analysis of these apolipoproteins isolated from serum HDL collected from a density gradient ultracentrifugation-based separation. The IPG separation of the HDL apolipoproteins was imaged and combined with digital analysis to produce a detailed pI profile of the apolipoproteins in the high-density lipoprotein (HDL) fraction. This is the first time that a high-resolution pI profile has been obtained for the HDL apolipoproteins. The feasibility of linking the pI profile to a MALDI-based molecular weight determination was achieved by incorporating passive elution of the intact proteins from the IPG gel with a four-component solvent mixture that solved the problem of recovery of the apolipoproteins from the IPG matrix. Twenty-five bands were observed in the pI profile. A survey analysis of 12 of these bands by MALDI indicated that they were associated with the known HDL apolipoproteins. While there is considerable overlap in the pI profiles of the apolipoproteins, linking the analysis with a MALDI-based second dimension in m/z is shown to be an efficient method to characterize this complex mixture of apolipoproteins.

Apolipoproteins isolated from human high density lipoprotein were analyzed by matrix-assisted laser desorption (MALDI) and electrospray ionization mass spectrometry (ESI-MS). The long-term objective of this study is to identify and characterize new apolipoproteins and isoforms for clinical studies incorporating some of the latest advances in analytical chemistry methodology including mass spectrometry. The focus of this paper is on developing an understanding of the link between MALDI and ESI-MS data in the analysis of this complex mixture of proteins. Forty-nine peaks were observed in the MALDI spectrum and 11 species in the ESI-MS spectrum. The MALDI spectra consisted of peaks corresponding to known apoA and apoC molecular ion adducts and 11 species that could not be identified. The ESI-MS data provided excellent confirmation of MW values for the prominent ions in the spectrum. New isoforms of apoA-I and apoA-II were observed corresponding to truncation of C-terminal neutral amino acids. A new kind of isoform of apoA-II was discovered where the double strand is cleaved in vivo and the single strand is cysteinylated at the cleavage site. Confirmation was obtained by studying the change in the MALDI spectrum after performic acid oxidation. Charge-state distributions in the ESI-MS spectra were used to confirm the identity of this new isoform. The charge state distribution in ESI-MS was found to correlate with the number of basic residues in the apolipoprotein.