Skip to main content

National Institutes of Health

Eunice Kennedy Shriver National Institute of Child Health and Human Development

2023 Annual Report of the Division of Intramural Research

NICHD Biomedical Mass Spectrometry Core Facility

Peter Backlund
  • Peter S. Backlund, PhD, Staff Scientist, Acting Director
  • Alfred L. Yergey, PhD, Scientist Emeritus

The NICHD Biomedical Mass Spectrometry Core Facility was created to provide high-end mass-spectrometric services to scientists within the NICHD Division of Intramural Research (DIR). Particular focus has been in the areas of proteomics, biomarker discovery, protein characterization, and detection of post-translational modifications. The Facility also performs quantitative analyses of small biomolecules, including lipids and steroids. In addition, the Facility develops and modifies methods for the isolation and detection of biomolecules by mass spectrometry, as well as novel methods for data analysis. The Facility is located in room 8S-261 of Building 10 on the NIH campus and serves both clinical and basic research laboratories within the NICHD intramural research program. As resources permit, we also collaborate with principal investigators (PIs) of other institutes within NIH and with other outside institutions.

The Facility is committed to promoting mass-spectrometric aspects of proteomics and other mass-spectrometric analyses in NICHD’s DIR. We provide advice and protocols for appropriate methods of sample isolation that are compatible with analysis by mass spectrometry. We also support an NIH–wide seminar series featuring experts in proteomics. In parallel, the staff of the Facility have developed collaborations with other Institutes to promote exchange of information and to bring new mass-spectrometric techniques to the NICHD. In addition, Peter Backlund is the moderator of the NIH Mass Spectrometry Interest Group.

Mode of operation

The Facility is available to all labs within the DIR, provided that existing resources are distributed equally among investigators requesting services. The Facility’s staff are available for consultation on both project design and data interpretation. Staff members meet with the PI and other scientists involved in each study to discuss experimental goals and data requirements. The Facility has capabilities in the characterization of proteins and peptides by mass spectrometry, including: (1) identification of proteins isolated by electrophoresis; (2) confirmation of molecular weights of recombinant or synthetic proteins and peptides; (3) determination of sites of specific post-translational modifications, including phosphorylation, glutamylation, AMPylation, and disulfide bond formation; (4) quantification of specific post-translational modifications; and (5) de novo sequencing of peptides. In addition, the Facility has extensive experience and skill in the identification and quantification of small endogenous molecules, including phospholipids, steroids, and sugars. In this latter area, the capability is primarily in quantification of endogenous levels of particular molecules and their metabolites.


The facility currently has four mass spectrometers in use for specific areas of analysis.


The state-of-the-art high-performance MALDI (matrix-assisted laser desorption/ionization) TOF/TOF (time-of-flight/time-of-flight) instrument can be operated in either positive- or negative-ion modes. The instrument is most often used for peptide identification in peptide mixtures without chromatographic separation. Methodology is also available to perform off-line liquid chromatography (LC) separation and sample spotting. Additional uses include relative peptide quantification for iTRAQ (isobaric tags for relative and absolute quantitation)–labeled peptides and sequence determination through de novo sequencing techniques for unusual peptides not present in gene-based protein databases.

Agilent 6560 Ion Mobility-qTOF

The state-of-the art instrument couples a one-meter ion-mobility-drift cell with a high-resolution qTOF mass spectrometer. Ion-mobility spectrometry (IMS) prior to mass analysis provides an added dimension of sample separation that is orthogonal to both chromatography and mass spectrometry. The instrument is currently used to determine collision cross-section measurements of ions for small molecules and intermolecular complexes and for separation and analysis of complex mixtures of lipids and peptides.

Agilent 6495 LC-ESI QqQ (Triple Quad)

The instrument is coupled to an Infinity 1290 UPLC (ultra-performance liquid chromatography) system with either an ESI (electrospray ionization) or APCI (atmospheric pressure chemical ionization) ion source, and is currently used for small-molecule analysis and quantification, principally for steroid profiling and the analysis of amino-acid and glycolytic-pathway metabolites.


The instrument is used for the analysis of protein mixtures and to verify molecular weights of intact proteins. It is also available for general use after a prospective user has undergone appropriate training.

Major projects

Ion-mobility mass spectrometry for detection of isobaric biomolecules and ion complexes

Given that ion-mobility spectrometry (IMS) operates on a millisecond time scale, the technique performs separations of complex mixtures much faster than is possible with liquid chromatography (LC). In addition, IMS separations are associated with the collision cross section (CCS) of ions (CCS is essentially a ‘shape’ parameter of ions in the gas phase), so that molecules of identical molecular weights but with different structures can be separated on the basis of their CCS. This has great potential for separating isobaric biomolecules, including numerous steroids, lipids, and peptides. In addition to the separation of structural isomers, IMS also offers the ability to study intermolecular complexes in the gas phase to determine conformational changes and stoichiometry. One of the first studies we undertook was to investigate beta-cyclodextrin–cholesterol complexes in the presence of various monovalent and divalent cations. The measured CCS of different ions could be attributed to two distinct conformations of the ions, and we have begun molecular modeling studies to independently explain the different conformational states. We also determined the reduced mobility (Ko) and CCS values for a group of analyte ions that had been previously characterized in other drift tube IM-MS instruments. In addition, we determined CCS values for both positively and negatively charged ions of cyclodextrins and maltodextrose [Reference 1]. The instrument was used to detect structural differences between two commercial preparations of hydroxypropyl-modified beta-cyclodextrins [Reference 2]. One of these preparations is currently being used in clinical trials to treat Neimann-Pick disease type C1 patients.

We are currently using IM-MS to analyze complex mixtures of phospholipids extracted from mouse tissues in order to analyze branched-chain fatty acid incorporation into phosphatidylcholine (PC) in animals fed a diet supplemented with phytol, a saturated C20 branched-chain alcohol. Phytol is metabolized to phytanic acid, which can be incorporated into phospholipids and triglycerides. The muscle PC species profiles under the phytol and control diet were similar, except that some additional species were detected in the phytol-diet muscle. We tentatively identified the two most abundant novel species as PC 20:0-16:0 and PC 20:0-22:6. The drift times for these novel species are consistent with the molecules containing the branched phytanoyl fatty acyl group. The separation of phospholipids by ion mobility also makes it possible to quantitate complex mixtures of PC species without the longer time period required for LC separation of these mixtures, shortening run times from 60 to 3 minutes of IMS separation.

The formation of PC complexes was also demonstrated using a mixture of two purified PCs, and we observed clusters of doubly charged positive ions ranging from trimers to octamers. The CCS values of the hepta- and octameric clusters show a significant departure from the trend line exhibited for trimers through hexamers, indicating a possible change in complex structure that may represent a transition to lipid self-organization.

Assay for quantitation of phosphatidyl-inositol mono-, di- and tri-phosphates

We implemented an LC-MRM (multiple reaction monitoring) assay method to measure endogenous levels of phosphatidyl inositol phosphates in tissues and virus particles. The method is being used to quantitate levels of these compounds in brain tissues and influenza virus particles.

Quantitation of plasma melatonin (5-methoxy-N-acetyltryptamine) and N-acetyltryptamine

We developed an MRM–based assay to quantify N-acetyltryptamine and melatonin in plasma. N-acetyltryptamine is a melatonin-receptor mixed agonist/antagonist. The assay provided the first evidence for endogenous N-acetyltryptamine in the daytime plasma from human volunteers, rhesus monkeys, and rats. The mass-spectrometric method employs deuterated internal standards to quantitate N-acetyltryptamine and melatonin. Twenty-four-hour studies of rhesus macaque plasma revealed elevations in N-acetyltryptamine at night to concentrations that exceed those of melatonin. We also used the technique to measure the compounds in tissues known to be involved in melatonin biosynthesis, and N-acetyltryptamine was present in both pineal and retinal tissue from rhesus macaques. The findings establish the physiological presence of N-acetyltryptamine in the circulation and support the hypothesis that the tryptophan metabolite plays a significant physiological role as an endocrine or paracrine chrono-biotic through actions mediated by the melatonin receptor [Reference 3].

Mass spectrometry–based profiling and quantification of serum and urinary steroids

We previously developed an MRM–based mass-spectrometry method to quantify several androgenic steroids in urine and applied the method to studies of polycystic ovary syndrome (PCOS) patients and patients with congenital adrenal hyperplasia (CAH). The assay was used to quantify 5-alpha-pregnane-3-alpha,17-alpha-diol-20-one (known also as pdiol) and its 5-beta stereoisomer, 17-alpha-hydroxypregnanolone (known also as 5-β-pdiol); pdiol is an intermediate in the ‘backdoor pathway’ from 17OH progesterone to dihydrotestosterone. In a study of CAH patients, we found urinary levels of both pdiol and 5-β-pdiol to be directly correlated with the serum levels of androstenedione. The assay also measures etiocholanolone, androsterone, and testosterone. More recently, we developed an MRM–based assay for quantification of glucocorticoids in serum, including cortisol, cortisone, 11-deoxycortisol, and corticosterone.


  1. Klein C, Cologna SM, Kurulugama RT, Blank PS, Darland E, Mordehai A, Backlund PS, Yergey AL. Cyclodextrin and malto-dextrose collision cross sections determined in a drift tube ion mobility mass spectrometer using nitrogen bath gas. Analyst 2018;143:4147-4154.
  2. Yergey AL, Blank PS, Cologna SM, Backlund PS, Porter FD, Darling AJ. Characterization of hydroxypropyl-beta-cyclodextrins used in the treatment of Niemann-Pick disease type C1. PLoS One 2017;12:e0175478.
  3. Backlund PS, Urbanski HF, Doll MA, Hein DW, Bozinoski M, Mason CE, Coon SL, Klein DC. Daily rhythm in plasma N-acetyltryptamine. J Biol Rhythms 2017;32:195-211.


  • Paul Blank, PhD, Section on Cellular and Membrane Biophysics, NICHD, Bethesda, MD
  • Stephanie M. Cologna, PhD, University of Illinois, Chicago, IL
  • Jens R. Coorssen, PhD, Brock University, St. Catharines, Ontario, Canada
  • David C. Klein, PhD, Section on Neuroendocrinology, NICHD, Bethesda, MD
  • Stephen H. Leppla, PhD, Laboratory of Parasitic Diseases, NIAID, Bethesda, MD
  • Joan Marini, MD, PhD, Section on Heritable Disorders of Bone and Extracellular Matrix, NICHD, Bethesda, MD
  • Deborah P. Merke, MD, MS, Pediatric Consult Service, NIH Clinical Center, Bethesda, MD
  • Matthew Olson, MD, The Mayo Clinics, Jacksonville, FL
  • Forbes Porter, MD, PhD, Section on Molecular Dysmorphology, NICHD, Bethesda, MD
  • Dan Sackett, PhD, Cytoskeletal Dynamics Group, NICHD, Bethesda, MD
  • Brian Searle, Proteome Software, Inc., Portland, OR
  • Stephen E. Stein, PhD, National Institute of Standards and Technology, Gaithersburg, MD
  • Gisela Storz, PhD, Section on Environmental Gene Regulation, NICHD, Bethesda, MD
  • Constantine A. Stratakis, MD, D(med)Sci, Section on Endocrinology and Genetics, NICHD, Bethesda, MD
  • Joshua Zimmerberg, MD, PhD, Section on Integrative Biophysics, NICHD, Bethesda, MD


For more information, email

Top of Page