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Longitudinal Analysis of Maternal Plasma Apolipoproteins in Pregnancy: A Targeted Proteomics Approach*

Open AccessPublished:October 10, 2012DOI:https://doi.org/10.1074/mcp.M112.018192
      Minimally invasive diagnostic tests are needed in obstetrics to identify women at risk for complications during delivery. The apolipoproteins fluctuate in complexity and abundance in maternal plasma during pregnancy and could be incorporated into a blood test to evaluate this risk. The objective of this study was to examine the relative plasma concentrations of apolipoproteins and their biochemically modified subtypes (i.e. proteolytically processed, sialylated, cysteinylated, dimerized) over gestational time using a targeted mass spectrometry approach. Relative abundance of modified and unmodified apolipoproteins A-I, A-II, C-I, C-II, and C-III was determined by surface-enhanced laser desorption/ionization-time of flight-mass spectrometry in plasma prospectively collected from 11 gravidas with uncomplicated pregnancies at 4–5 gestational time points per patient. Apolipoproteins were readily identifiable by spectral pattern. Apo C-III2 and Apo C-III1 (doubly and singly sialylated Apo C-III subtypes) increased with gestational age (r2>0.8). Unmodified Apo A-II, Apo C-I, and Apo C-III0 showed no correlation (r2 = 0.01–0.1). Pro-Apo C-II did not increase significantly until third trimester (140 ± 13% of first trimester), but proteolytically cleaved, mature Apo C-II increased in late pregnancy (702 ± 130% of first trimester). Mature Apo C-II represented 6.7 ± 0.9% of total Apo C-II in early gestation and increased to 33 ± 4.5% in third trimester. A label-free, semiquantitative targeted proteomics approach was developed using LTQ-Orbitrap mass spectrometry to confirm the relative quantitative differences observed by surface-enhanced laser desorption/ionization-time of flight-mass spectrometry in Apo C-III and Apo C-II isoforms between first and third trimesters. Targeted apolipoprotein screening was applied to a cohort of term and preterm patients. Modified Apo A-II isoforms were significantly elevated in plasma from mothers who delivered prematurely relative to term controls (p = 0.02). These results support a role for targeted proteomics profiling approaches in monitoring healthy pregnancies and assessing risk of adverse obstetric outcomes.
      The maternal physiology during pregnancy is characterized by inflammation and hyperlipidemia. Plasma protein composition fluctuates dynamically throughout gestation to reflect these physiological changes. Apolipoproteins, a diverse subset of triglyceride transport proteins, contribute to the hyperlipidemia of pregnancy by modulating lipid homeostasis in maternal plasma (
      • Aouizerat B.E.
      • Kulkarni M.
      • Heilbron D.
      • Drown D.
      • Raskin S.
      • Pullinger C.R.
      • Malloy M.J.
      • Kane J.P.
      Genetic analysis of a polymorphism in the human apoA-V gene: effect on plasma lipids.
      ,
      • Mahley R.W.
      • Innerarity T.L.
      • Rall Jr., S.C.
      • Weisgraber K.H.
      Plasma lipoproteins: apolipoprotein structure and function.
      ,
      • Montes A.
      • Knopp R.H.
      Lipid metabolism in pregnancy. IV. C Apoprotein changes in very low and intermediate density lipoproteins.
      ). Exaggerated hyperlipidemia and peripheral apolipoprotein burden are associated with inflammatory insult and signal obstetric complications (
      • Nagy B.
      • Rigó Jr., J.
      • Fintor L.
      • Romics L.
      • Papp Z.
      • Karádi I.
      Distribution of apolipoprotein(a) isoforms in normotensive and severe preeclamptic women.
      ,
      • Redman C.
      • Sargent I.L.
      Immunological factors and placentation: implications for preeclampsia.
      ). Numerous post-translationally modified apolipoprotein isoforms are reported in plasma, but it is unclear how these modifications affect apolipoprotein function and plasma distribution. For example, changes in the glycosylation status of apolipoprotein variants predate the onset of clinical symptoms in patients with preeclampsia, a hypertensive disorder of pregnancy with clinical features in common with cardiovascular disease (
      • Atkinson K.R.
      • Blumenstein M.
      • Black M.A.
      • Wu S.H.
      • Kasabov N.
      • Taylor R.S.
      • Cooper G.J.
      • North R.A.
      An altered pattern of circulating apolipoprotein E3 isoforms is implicated in preeclampsia.
      ,
      • Blumenstein M.
      • McMaster M.T.
      • Black M.A.
      • Wu S.
      • Prakash R.
      • Cooney J.
      • McCowan L.M.
      • Cooper G.J.
      • North R.A.
      A proteomic approach identifies early pregnancy biomarkers for preeclampsia: novel linkages between a predisposition to preeclampsia and cardiovascular disease.
      ,
      • Flood-Nichols S.
      • Stallings J.D.
      • Gotkin J.L.
      • Tinnemore D.T
      • Napolitano P.N
      • Ippolito D.L.
      Elevated Ratio of Maternal Plasma Ratio of ApoCIII to ApoCII in Preeclampsia.
      ). The identification and functional characterization of plasma apolipoprotein isoforms and their post-translationally modified subtypes may reveal important diagnostic and/or therapeutic targets for hypertensive disorders of pregnancy (
      • Atkinson K.R.
      • Blumenstein M.
      • Black M.A.
      • Wu S.H.
      • Kasabov N.
      • Taylor R.S.
      • Cooper G.J.
      • North R.A.
      An altered pattern of circulating apolipoprotein E3 isoforms is implicated in preeclampsia.
      ).
      Mass spectrometry and targeted proteomics analyses afford unprecedented sensitivity and specificity for detecting apolipoproteins and their numerous isoforms and subtypes (
      • Levels J.H.
      • Bleijlevens B.
      • Rezaee F.
      • Aerts J.M.
      • Meijers J.C.
      SELDI-TOF mass spectrometry of High-Density Lipoprotein.
      ,
      • Nelsestuen G.L.
      • Zhang Y.
      • Martinez M.B.
      • Key N.S.
      • Jilma B.
      • Verneris M.
      • Sinaiko A.
      • Kasthuri R.S.
      Plasma protein profiling: unique and stable features of individuals.
      ,
      • Shankar R.
      • Cullinane F.
      • Brennecke S.P.
      • Moses E.K.
      Applications of proteomic methodologies to human pregnancy research: a growing gestation approaching delivery?.
      ,
      • Shankar R.
      • Gude N.
      • Cullinane F.
      • Brennecke S.
      • Purcell A.W.
      • Moses E.K.
      An emerging role for comprehensive proteome analysis in human pregnancy research.
      ). Mass spectrometry approaches overcome limitations inherent in biochemical approaches (e.g. ELISA [enzyme-linked immunosorbant assays] and Western blot analysis), especially the lack of specificity of antibodies for post-translationally modified variants of plasma proteins. The objective of this study was to longitudinally evaluate maternal plasma apolipoprotein profile over gestational time by SELDI-TOF-MS (surface-enhanced laser desorption/ionization-time of flight-mass spectrometry)
      The abbreviations used are:
      MS/MS
      tandem mass spectrometry
      Apo C-I'
      Apo C-I without N-terminal threonine-proline
      Apo C-III2
      di-sialylated Apo C-III
      Apo C-III1
      mono-sialylated Apo C-III
      Apo C-III0
      a-sialylated Apo C-III
      Apo A-II'
      cysteinylated Apo A-II without C-terminal glutamine
      Apo A-IId
      dimeric cysteinylated Apo A-II minus two C-terminal glutamine residues
      Apo A-IId'
      dimeric cysteinylated Apo C-II minus one C-terminal glutamine
      Apo A-IId
      dimeric Apo A-II
      H50
      hydrophobic arrays
      HDL
      high density lipoproteins
      SRM
      selected reaction monitoring
      MRM
      multiple reaction monitoring
      ACOG
      American College of Obstetrics and Gynecology
      GalNAc
      N-acetylgalactosamine
      Gal
      galactose
      FDR
      false discovery rate
      LDL
      low-density lipoproteins
      VLDL
      very low-density lipoproteins
      LTQ
      linear trap quadrupole
      AUC
      area under the extracted ion chromatogram
      LH
      luteinizing hormone
      CID
      collision-induced dissociation
      MALDI-TOF-MS
      matrix-assisted laser desorption/ionization-time of flight-mass spectrometry
      SELDI-TOF-MS
      surface-enhanced laser desorption/ionization-time of flight-mass spectrometry.
      1The abbreviations used are:MS/MS
      tandem mass spectrometry
      Apo C-I'
      Apo C-I without N-terminal threonine-proline
      Apo C-III2
      di-sialylated Apo C-III
      Apo C-III1
      mono-sialylated Apo C-III
      Apo C-III0
      a-sialylated Apo C-III
      Apo A-II'
      cysteinylated Apo A-II without C-terminal glutamine
      Apo A-IId
      dimeric cysteinylated Apo A-II minus two C-terminal glutamine residues
      Apo A-IId'
      dimeric cysteinylated Apo C-II minus one C-terminal glutamine
      Apo A-IId
      dimeric Apo A-II
      H50
      hydrophobic arrays
      HDL
      high density lipoproteins
      SRM
      selected reaction monitoring
      MRM
      multiple reaction monitoring
      ACOG
      American College of Obstetrics and Gynecology
      GalNAc
      N-acetylgalactosamine
      Gal
      galactose
      FDR
      false discovery rate
      LDL
      low-density lipoproteins
      VLDL
      very low-density lipoproteins
      LTQ
      linear trap quadrupole
      AUC
      area under the extracted ion chromatogram
      LH
      luteinizing hormone
      CID
      collision-induced dissociation
      MALDI-TOF-MS
      matrix-assisted laser desorption/ionization-time of flight-mass spectrometry
      SELDI-TOF-MS
      surface-enhanced laser desorption/ionization-time of flight-mass spectrometry.
      analysis of intact proteins and a complementary targeted LTQ-Orbitrap XL MS approach. We evaluate changes in 13 post-translationally modified subtypes of the plasma apolipoproteins A-II, C-I, C-II, and C-III over gestational time.

      DISCUSSION

      In this study, we use SELDI-TOF-MS and LTQ-Orbitrap targeted proteomics to isolate and semiquantitatively characterize post-translationally modified subtypes of plasma apolipoproteins throughout gestation. We demonstrate the novel findings that a mature, cleaved form of Apo C-II increases over gestational time, and provide evidence that a panel of apolipoprotein subtypes may be useful in predicting preterm birth.
      To our knowledge, this study is the first to report a disproportionate increase in mature Apo C-II relative to pro-Apo C-II subtypes in pregnancy using the detection capabilities unique to mass spectrometry. Studies using SELDI-TOF-MS have reported the increase in abundance of an 8204 Da peak coincident with mature Apo C-II in clinical conditions associated with increased inflammation. Systemic IL-2 administration resulted in an increase in the 8204 Da Apo C-II peak, suggesting a correlation between mature Apo C-II and inflammatory insult (
      • Rossi L.
      • Martin B.M.
      • Hortin G.L.
      • White R.L.
      • Foster M.
      • Moharram R.
      • Stroncek D.
      • Wang E.
      • Marincola F.M.
      • Panelli M.C.
      Inflammatory protein profile during systemic high dose interleukin-2 administration.
      ). In normal physiology, only about 5% of Apo C-II exists in adult plasma in its cleaved, mature form (lacking the N-terminal TQQPQQ sequence) (
      • Fojo S.S.
      • Taam L.
      • Fairwell T.
      • Ronan R.
      • Bishop C.
      • Meng M.S.
      • Hoeg J.M.
      • Sprecher D.L.
      • Brewer Jr., H.B.
      Human preproapolipoprotein C-II. Analysis of major plasma isoforms.
      ). Pro-Apo C-II represents the remaining 95%. Mass spectrometry-based studies confirm a proportionally smaller quantity of mature Apo C-II in plasma relative to pro-Apo-CII (
      • Timms J.F.
      • Arslan-Low E.
      • Gentry-Maharaj A.
      • Luo Z.
      • T'Jampens D.
      • Podust V.N.
      • Ford J.
      • Fung E.T.
      • Gammerman A.
      • Jacobs I.
      • Menon U.
      Preanalytic influence of sample handling on SELDI-TOF serum protein profiles.
      ).
      ELISA and immunoturbidimetric assays show that Apo A-I, C-I, and A-II increase proportionally between first and second trimesters (
      • Montes A.
      • Knopp R.H.
      Lipid metabolism in pregnancy. IV. C Apoprotein changes in very low and intermediate density lipoproteins.
      ,
      • Alvarez J.J.
      • Montelongo A.
      • Iglesias A.
      • Lasunción M.A.
      • Herrera E.
      Longitudinal study on lipoprotein profile, high density lipoprotein subclass, and postheparin lipases during gestation in women.
      ,
      • Mazurkiewicz J.C.
      • Watts G.F.
      • Warburton F.G.
      • Slavin B.M.
      • Lowy C.
      • Koukkou E.
      Serum lipids, lipoproteins and apolipoproteins in pregnant non-diabetic patients.
      ,
      • Piechota W.
      • Staszewski A.
      Reference ranges of lipids and apolipoproteins in pregnancy.
      ,
      • Roy A.C.
      • Loke D.F.
      • Saha N.
      • Viegas O.A.
      • Tay J.S.
      • Ratnam S.S.
      Interrelationships of serum paraoxonase, serum lipids and apolipoproteins in normal pregnancy. A longitudinal study.
      ). As expected in pregnancy, we observe a significant decrease in ratios of Apo A-I:Apo A-II and Apo A-IIdimer subtypes from trimester 1 to trimester 3, and an appreciable increase in VLDL/LDL cholesterol but not HDL cholesterol. Immunological methods lack sufficient sensitivity to account for variation in apolipoprotein subtypes in pregnancy. The discrepancy between the SELDI-TOF-MS and ELISA data could also be explained by competitive binding of proteins present in high molar ratios to the array surfaces. The changes in subtypes reported in this study warrant further investigation in larger cohorts of complicated pregnancies.
      Caution must be exercised when comparing proteomic profiles in physiological states such as pregnancy which are characterized by dynamic changes in plasma protein complement. Because SELDI-TOF-MS arrays preferentially bind proteins of greater molar abundance, significant changes in protein complement can artificially affect intensities of other spectral features (
      • Hortin G.L.
      The MALDI-TOF mass spectrometric view of the plasma proteome and peptidome.
      ). This limitation of the SELDI-TOF-MS technique may be overcome by fractionating specimens to limit the competitive inhibition of high molar abundance species in maternal plasma (
      • De Bock M.
      • de Seny D.
      • Meuwis M.A.
      • Chapelle J.P.
      • Louis E.
      • Malaise M.
      • Merville M.P.
      • Fillet M.
      Challenges for biomarker discovery in body fluids using SELDI-TOF-MS.
      ). However, one of the limitations of most of the mass spectrometry techniques described in this study is the ability to detect only relative changes in abundance. The concentrations of Apo C-III calculated by interpolation to an internal standard are lower than predicted by ELISA (an average difference of 290 μg/ml, Supplemental Table S4), suggesting that the sialyl groups caused significant attenuation of ionization in the SELDI-TOF technique. During laser desorption in mass spectrometry, variable loss of sialic acid occurs in proportion to laser energy and other factors. From a biochemical standpoint, the ELISA antibodies detect all subtypes of Apo C-III, whereas our mass spectrometry techniques examine the subtypes individually. Therefore, both the biochemical and mass spectrometry approaches have inherent limitations making it difficult to obtain accurate quantitation of individual subtypes in a given sample.
      Bondarenko and colleagues demonstrated the utility of electrospray mass spectrometry in characterizing apolipoprotein subtypes by analyzing intact proteins (
      • Bondarenko P.V.
      • Cockrill S.L.
      • Watkins L.K.
      • Cruzado I.D.
      • Macfarlane R.D.
      Mass spectral study of polymorphism of the apolipoproteins of very low density lipoprotein.
      ,
      • Niederkofler E.E.
      • Tubbs K.A.
      • Kiernan U.A.
      • Nedelkov D.
      • Nelson R.W.
      Novel mass spectrometric immunoassays for the rapid structural characterization of plasma apolipoproteins.
      ). More recently, novel fucosylated Apo C-III subtypes were identified by mass spectrometry analysis of Schistosoma mansoni from urine of infected individuals (
      • Balog C.I.
      • Mayboroda O.A.
      • Wuhrer M.
      • Hokke C.H.
      • Deelder A.M.
      • Hensbergen P.J.
      Mass spectrometric identification of aberrantly glycosylated human apolipoprotein C-III peptides in urine from Schistosoma mansoni-infected individuals.
      ). Serum isoforms of Apo C-I, Apo C-II, Apo C-III, Apo A-II, dimerized Apo A-II, and Apo A-I were predictive markers for Alzheimers disease in MALDI-TOF-MS studies (
      • Niederkofler E.E.
      • Tubbs K.A.
      • Kiernan U.A.
      • Nedelkov D.
      • Nelson R.W.
      Novel mass spectrometric immunoassays for the rapid structural characterization of plasma apolipoproteins.
      ).
      Although we acknowledge that our findings of changes in concentration for dimerized Apo A-II in preterm patients must be validated in a larger cohort, our results warrant further investigation into the use of apolipoprotein subtype analysis in obstetric populations. Shotgun proteomics strategies in obstetric cohorts have yielded some success in biomarker identification in blood for early diagnosis of pregnancy complications (
      • Atkinson K.R.
      • Blumenstein M.
      • Black M.A.
      • Wu S.H.
      • Kasabov N.
      • Taylor R.S.
      • Cooper G.J.
      • North R.A.
      An altered pattern of circulating apolipoprotein E3 isoforms is implicated in preeclampsia.
      ,
      • Blumenstein M.
      • McMaster M.T.
      • Black M.A.
      • Wu S.
      • Prakash R.
      • Cooney J.
      • McCowan L.M.
      • Cooper G.J.
      • North R.A.
      A proteomic approach identifies early pregnancy biomarkers for preeclampsia: novel linkages between a predisposition to preeclampsia and cardiovascular disease.
      ,
      • Choolani M.
      • Narasimhan K.
      • Kolla V.
      • Hahn S.
      Proteomic technologies for prenatal diagnostics: advances and challenges ahead.
      ,
      • Dasari S.
      • Pereira L.
      • Reddy A.P.
      • Michaels J.E.
      • Lu X.
      • Jacob T.
      • Thomas A.
      • Rodland M.
      • Roberts Jr., C.T.
      • Gravett M.G.
      • Nagalla S.R.
      Comprehensive proteomic analysis of human cervical-vaginal fluid.
      ,
      • Gravett M.G.
      • Thomas A.
      • Schneider K.A.
      • Reddy A.P.
      • Dasari S.
      • Jacob T.
      • Lu X.
      • Rodland M.
      • Pereira L.
      • Sadowsky D.W.
      • Roberts Jr., C.T.
      • Novy M.J.
      • Nagalla S.R.
      Proteomic analysis of cervical-vaginal fluid: identification of novel biomarkers for detection of intra-amniotic infection.
      ,
      • Michaels J.E.
      • Dasari S.
      • Pereira L.
      • Reddy A.P.
      • Lapidus J.A.
      • Lu X.
      • Jacob T.
      • Thomas A.
      • Rodland M.
      • Roberts Jr., C.T.
      • Gravett M.G.
      • Nagalla S.R.
      Comprehensive proteomic analysis of the human amniotic fluid proteome: gestational age-dependent changes.
      ,
      • Nagalla S.R.
      • Canick J.A.
      • Jacob T.
      • Schneider K.A.
      • Reddy A.P.
      • Thomas A.
      • Dasari S.
      • Lu X.
      • Lapidus J.A.
      • Lambert-Messerlian G.M.
      • Gravett M.G.
      • Roberts Jr., C.T.
      • Luthy D.
      • Malone F.D.
      • D'Alton M.E.
      Proteomic analysis of maternal serum in down syndrome: identification of novel protein biomarkers.
      ,
      • Pereira L.
      • Reddy A.P.
      • Jacob T.
      • Thomas A.
      • Schneider K.A.
      • Dasari S.
      • Lapidus J.A.
      • Lu X.
      • Rodland M.
      • Roberts Jr., C.T.
      • Gravett M.G.
      • Nagalla S.R.
      Identification of novel protein biomarkers of preterm birth in human cervical-vaginal fluid.
      ,
      • Rasanen J.
      • Girsen A.
      • Lu X.
      • Lapidus J.A.
      • Standley M.
      • Reddy A.
      • Dasari S.
      • Thomas A.
      • Jacob T.
      • Pouta A.
      • Surcel H.M.
      • Tolosa J.E.
      • Gravett M.G.
      • Nagalla S.R.
      Comprehensive maternal serum proteomic profiles of preclinical and clinical preeclampsia.
      ), but most of these studies require prefractionation and depletion of the most abundant proteins in plasma. These preprocessing modifications risk concomitant depletion of lower abundance proteins. Acetonitrile precipitation of high abundance proteins may account for the discrepancies between the SELDI-TOF-MS and Orbitrap data in our study, particularly among the Apo A-II and Apo C-I subtypes. Further, we did not investigate differences of 2 atomic mass units among our discriminatory peptides to differentiate monomeric from dimeric Apo A-II. Our quantification methods using 10 pmol of internal standard AQUA peptides (Orbitrap) or protein standard (SELDI-TOF-MS) are also limited in that variable loss of sialic acid which could result from laser desorption (in the case of SELDI-TOF-MS), depending on factors such as the laser energy used (
      • Hortin G.L.
      The MALDI-TOF mass spectrometric view of the plasma proteome and peptidome.
      ). Thus, our quantification probably underestimates the true content of Apo C-III in plasma. Alternatively, the antibody used in the ELISA is not specific for multiple apolipoprotein subtypes. The elevated concentration reported in the ELISA relative to the SELDI-TOF-MS quantification could reflect greater specificity of the mass spectrometry approach in detecting unique subtypes.
      In this study, we evaluate LTQ-Orbitrap XL mass spectrometry as a complementary experimental approach to estimate changes in apolipoprotein subtype abundance. Our approach relies on determining the area under the extracted ion chromatogram of differentiable peptides, thus allowing for discrimination between apolipoprotein subtypes. The accurate mass capability of the Orbitrap renders this level of specificity possible, allowing for improved differentiation among the subtypes in the Orbitrap platform.
      In conclusion, this study demonstrates the utility of a SELDI-TOF-MS proteomic profiling strategy to (1) detect apolipoprotein subtypes in plasma with minimal sample manipulation and (2) observe changes in protein profile in an obstetrically relevant biological problem (prematurity). The LTQ-Orbitrap XL relative quantification and targeted identification of modified peptides provide a complementary experimental approach to SELDI-TOF-MS for the Apo C-III, Apo C-II, and Apo A-II subtypes investigated. This study reports novel evidence for the presence and fluctuation of modified isoforms of apolipoproteins in maternal plasma. In particular, we describe for the first time an increase in mature Apo C-II relative to pro-Apo C-II over gestational time. Because database searching algorithms frequently do not incorporate post translational modifications of apolipoprotein isoforms, this study provides important evidence that many isoforms of proteins might be overlooked in conventional shotgun proteomics approaches.

      Acknowledgments

      We thank all mothers who volunteered for this project. We thank Patrick McNutt, PhD, for contributing to the conception of the Pregnancy Proteome Project; the Madigan OB-GYN nursing staff for recruiting patients; and the Madigan Pathology Laboratory for phlebotomy. We thank Phil Gafken, PhD, for LTQ-Orbitrap XL consultation and experimentation. We gratefully acknowledge the contributions of Mariano Mesngon, PhD, in manuscript review and figure generation. We thank David McCune, MD, and Richard Burney, MS, MD, for scientific and clinical guidance. We thank Troy Patience and Raywin Huang, PhD, for computational and statistical support, and Cindy Kirker for library assistance. D.L.I., D.T., and S.K.F.-N. jointly conceived of the study; D.L.I., D.T., S.K.F.-N., M.A.W., and A.I.A.-A. designed and performed the experiments; S.K.F.-N. reviewed patient antepartum medical records; D.L.I., J.D.S., and D.T. analyzed the data and interpreted the results; D.L.I. and D.T. wrote the paper; D.L.I., P.G.N., and J.D.S. supervised the project.

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