TABLE II

Secondary reverse-phase elution map of intact mass tags from appressed-membrane subfractions of pea thylakoids (n = 2)

IMTa mass (± range)Calculated massesbΔcIDdModificationse
20261.3
22233.6*22233.8Lhca?
22841.1*22842.5Lhcb6NH2-A??
23345.0 ± 0.2*23345.8Lhca?
24346.9 ± 0.524330.8 1 Lhcb3NH2-G43
24751.2 ± 0.5*24750.7Lhcb1/2Ac-R??
24838.3 ± 0.124837.1XLhcb2Ac-R38
24958.624956.3XLhcb1*2Ac-R38
24972.224972.3XLhcb1*4Ac-R28
24981.2 ± 0.2*24981.1Lhcb1/2Ac-R??
25036.125036.3XLhcb1*2Ac-R38, HPO3-T40
25061.4$25061.1Lhcb1/2Ac-R??, HPO3-T?? 26563.5 ± 0.5*26562.8“Lhcb5”NH2-? 26526.9 27379.2*27379.2 28691.7 ± 0.2*28690.8“Lhcb4”Ac-? 9283.39283.4XPsbE 55895.1 ± 0.255902.0YPsbBAc-G2 17919.2 ± 0.1a 221678.6 ± 1.6*22169.8XPsbSAc 38031.4 ± 1.638033.6XPsbA.1fAc-T2, A344-COOH 38111.138113.6XPsbA.1fAc-T2, HPO3-T2, A344-COOH 38040.738040.9XPsbA.2fAc-T2, A344-COOH 38121.338120.9XPsbA.2fAc-T2, HPO3-T2, A344-COOH 50201.4 ± 5.650204.9XPsbCAc-T15 50285.050284.9XPsbCAc-T15, HPO3-T15 4366.2 ± 1.64366.0XPsbLNH2-T2 39444.2 ± 3.239437.5XPsbD 39487.039479.5YPsbDAc-T2 39520.739517.5XPsbD 39550.039559.5ZPsbDAc-T2, HPO3-T2 4026.0 ± 1.64026.7XPsbJAc-A2 4157.3f 4174.3f+O 4060.5 ± 0.34032.5 2 PsbTfM 4141.5 ± 0.14112.5 2 PsbTfM, + HPO3 4209.8 ± 0.14209.8XPsbIfM 3782.93783.6YPsbMfM 6641.5 ± 0.16582.8 3 Ycf9fM • a IMTs assembled in order of elution during the secondary reverse-phase chromatographic elution. Grouped IMTs indicate co-elution. IMTs without a range were observed in only one or the other experiment. • b Calculated average mass of the uncharged assigned gene product shown in ID column (Da). *, mass observed in Table I but with no published gene sequence from which to predict a mass.$, modification of a (*) mass observed in Table I.

• c Δ, % difference between expected and observed masses; X, Δ < 0.01%; Y, Δ = 0.01–0.02%; Z, Δ = 0.02–0.07%. 1, the mass and relative elution time of IMT 24347.4 Da is consistent with Lhcb3. The difference may be because of one of several possible single amino acid substitutions. 2, the published pea PsbT gene sequence appears to have an in-frame deletion (see text). 3, the IMT 6641.5 Da elutes at a time consistent with that predicted for Ycf9 (a conserved protein in plant chloroplast genomes of unknown function) and has similar mass. The discrepancy may be because of published errors in the DNA sequence or because of allelic differences in the cultivars used (Alaska, this study; Rosakrona, M27309).

• d Protein IDs in quotes indicate assignments made by retention times consistent with membrane localization and similar masses from published sequences from other plants.

• e O, oxygen; Ac, acetyl; fM, N-formylmethionine; HPO3, O-phosphorylation. Two unidentified masses that are observed to be modified are labeled in lower case letters (a, f). Superscripts indicate position from Met as amino acid 1. Sequence accession numbers are listed in the footnotes to Table I, except for Ycf9 (M27309).

• f In this experiment (Pisum sativum cv. Alaska) there are potentially two isoforms of D1. The first isoform (38031.4 Da) is within 0.006% of the mass calculated from the published sequence (M11005). The second isoform (38040.7 Da) is within 0.0004% of the mass observed in previous LC-MS experiments (15). Both isoforms are consistent with removal of fMet, acetylation of residue 2, and C-terminal removal of the last nine amino acids, and both have phosphorylated subpopulations. There are several single amino acid substitutions that could account for the 7-Da difference in isoform masses. Further experiments using various pea cultivars are necessary to confirm this observation.