RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom. 2003; 17: 2226–2232 Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rcm.1179 Determination of phosphorylation sites in lipooligosaccharides from Pseudoalteromonas haloplanktis TAC 125 grown at 158C and 258C by nano-electrospray ionization quadrupole time-of-flight tandem mass spectrometry S. Ummarino 1 , M. M. Corsaro 1 *, R. Lanzetta 1 , M. Parrilli 1 and J. Peter-Katalinic ´ 2 1 Dipartimento di Chimica Organica e Biochimica, Universita ` Federico II di Napoli, Complesso Universitario Monte S. Angelo, Via Cynthia 4, 80126 Napoli, Italy 2 Institute for Medical Physics and Biophysics, University of Mu ¨nster, 48149 Mu ¨nster, Germany Received 22 July 2003; Revised 6 August 2003; Accepted 6 August 2003 Lipooligosaccharides (LOSs) are macromolecules present on the external cellular membrane of Gram-negative bacteria, structurally made of two distinct regions, lipid A and Core. By varying their growth temperature, bacteria such as psychrophiles change the phosphorylation distribution of the LOSs produced. The level of phosphorylation and the phosphate group positions in LOSs produced by the extremophile psychrophilic bacterium Pseudoalteromonas haloplanktis TAC 125, grown at 158C and 258C, were investigated by nano-electrospray ionization quadrupole time-of- flight mass spectrometry (nanoESI-QTOF-MS) and tandem mass spectrometry (MS/MS). The sam- ples, obtained by phenol/chloroform/petroleum ether (PCP) extraction of dried cells, were treated with hydrazine at 378C in order to reduce the heterogeneity by removal of the ester-linked fatty acid moieties. The molecular ion distributions in these LOS fractions were investigated in negative ion mode. Based on these data it was postulated that the sample grown at 258C contained four phos- phate groups while that at 158C contained three. In order to determine phosphorylation sites in sugar chains, the samples were submitted to low collision energy MS/MS for sequencing. In the sample with three phosphates, one was found to be linked to the tetrasaccharide Core region, more precisely to position C-4 of the Kdo unit. The two remaining phosphate groups were both linked to the 2-acylamide-2-deoxy-D-glucopyranose of the lipid A moiety, and two possible distri- butions could be postulated on the basis of the fragmentation pattern obtained; in the first case both phosphate groups are linked as a pyrophosphate moiety to position C-1 of the proximal glu- cosamine (reducing residue), while in the second case one phosphate is linked to position C-1 of the proximal glucosamine and the other to position C-4 0 of the distal glucosamine (non-reducing residue). This distribution was also found in the lipid A moiety of the tetraphosphorylated sample grown at 258C, which bears two phosphate groups on the Core region, one on position C-4 of the Kdo and the other on position C-7 or C-8 of the same residue. The phosphate locations were derived from the intra-ring cleavage ions of sugar moieties in the LOSs obtained by an optimized CID procedure using negative ion QTOF-MS/MS. Copyright # 2003 John Wiley & Sons, Ltd. Psychrophilic bacteria are microorganisms which live under low-temperature conditions, generally below 58C. In order to survive and grow in cold environments they have evolved via a complex range of adaptations to all of their cellular and extra-cellular components. Most interest in this respect has been addressed to the protein and lipid components. 1,2 Much less attention has been paid to the lipopolysaccharide (LPS) component which, being located on the periphery of the external membrane of Gram-negative bacteria, is in the closest proximity to the outside environment. Lipopolysaccharides consist of three distinct regions, namely, the lipid A moiety, the Core oligosaccharide, and the O-antigen polysaccharide. 3 The lipid A moiety, which serves as the hydrophobic anchor of the LPS in the outer membrane, represents the endotoxic principle of LPS. Chemically, lipid A usually consists of a b-(1 ! 6) linked Copyright # 2003 John Wiley & Sons, Ltd. *Correspondence to: M. M. Corsaro, Dipartimento di Chimica Organica e Biochimica, Universita ` Federico II di Napoli, Complesso Universitario Monte S. Angelo, Via Cynthia 4, 80126 Napoli, Italy. E-mail: corsaro@unina.it Contract/grant sponsor: Ministero dell’Universita ` e della Ricerca Scientifica.