TABLE I

Generalized primary reverse-phase elution map of intact mass tags from appressed thylakoid membrane subfractions

IMTa pea (± S.D.) (n = 4 experiments)Calculated massesb (Da)ΔcIMTa spinach (± S.D.) (n = 7 experiments)Calculated massesb (Da)ΔcIDd
3296.5 ± 0.23297.9ZPsbX + O
3187.3 ± 0.23279.7 ± 0.43281.9ZPsbX
3485.2 ± 0.43163.2 ± 0.3
3000.0 ± 0.7
2088.7 ± 0.2
9894.3 ± 1.29728.9 ± 0.99729.0XPsaE
9824.4 ± 1.19657.7 ± 2.09657.9XPsaEΔAla
4028.8 ± 0.5
9721.3 ± 0.9“PsaN”
9150.5 ± 1.3
8641.0 ± 1.6
8848.4 ± 0.98849.3XPsaC
17936.3 ± 2.317871.8 ± 1.417872.6XPsaD + O
17921.3 ± 1.417856.7 ± 2.117856.6XPsaD
20225.4 ± 1.520225.6XPsbP + O
20265.0 ± 1.620264.6X20209.6 ± 2.020209.6XPsbP
16375.2 ± 2.016522.7 ± 2.916521.8XPsbQ
17829.4 ± 2.1a + 2F
17801.7 ± 2.7a + F
17774.6 ± 2.3a
16143.4 ± 2.6b + Ac
16102.0 ± 1.0b
12361.4 ± 1.3
26525.0 ± 1.826525.7X26532.1 ± 2.926661.8 1 PsbO
10276.3 ± 0.910381.6 ± 1.210381.8XPsaH
15564.7 ± 2.0
12941.4 ± 2.5
13815.4 ± 1.8
22037.2 ± 2.0
18956.5 ± 3.218954.6XPetC + O
19074.7 ± 2.419074.8X18940.4 ± 2.318938.6XPetC
11320.3 ± 1.310764.8 ± 0.5
29474.1 ± 2.6
8596.3 ± 0.8
6927.9 ± 1.36924.0 ± 1.1c + Ac + O
6911.6 ± 0.76907.3 ± 0.9c + Ac
6869.2 ± 0.26869.0 ± 1.5c
4411.1 ± 1.24411.2X4424.7 ± 0.54425.2XPsbF + O
4394.9 ± 0.34395.2X4409.2 ± 0.64409.2XPsbF
15178.6 ± 1.015171.8 ± 2.4
22230.6 ± 1.8
21781.0 ± 1.8d + Ac
21677.3 ± 1.6d + O
21739.0 ± 1.321661.2 ± 3.6d
21441.8 ± 2.8
4854.8 ± 0.7
25285.3 ± 3.325298.6 ± 2.9
10205.7 ± 1.410234.9 ± 1.410235.7X PsbR
8389.0 ± 0.49685.3 ± 0.9
21782.7 ± 2.822345.6 ± 2.5
17255.3 ± 1.217272.3 ± 3.017275.1YPsaF
3983.3 ± 0.3
4585.6 ± 0.6
24574.8 ± 1.324839.7 ± 2.8
24972.0 ± 2.224972.3X24760.2 ± 2.3Ac-Lhcb1*Ps4
24981.1 ± 1.925019.1 ± 3.425020.4XAc-Lhcb1*So1
25036.4 ± 2.225036.3XAc-Lhcb1*Ps2 + P
24959.1 ± 1.524956.3Y24946.3 ± 2.9Ac-Lhcb1*Ps2
24836.4 ± 2.124837.1XAc-Lhcb2*Ps1
24750.7 ± 1.924936.2 ± 2.6
24839.1 ± 1.425083.7 ± 3.0
24774.2 ± 1.925005.2 ± 3.2
22913.4 ± 1.622856.1 ± 3.4
22842.5 ± 1.622814.0 ± 1.922813.0XLhcb6*So1
22302.9 ± 1.8
22233.8 ± 1.6
24332.7 ± 2.324330.8X24323.3 ± 2.3Lhcb3*Ps1
22952.9 ± 3.1
24345.9 ± 2.0
28690.8 ± 2.328071.8 ± 3.9“Lhcb4”
26262.5 ± 2.4
26562.8 ± 1.927067.4 ± 2.7“Lhcb5”
22169.8 ± 1.822568.6 ± 2.122568.3XAc-PsbS
4540.3 ± 0.44960.4 ± 0.54960.8XPsbY2
4482.0 ± 0.0“PsbK”
23345.8 ± 2.023324.0 ± 2.9
27379.2 ± 2.623279.4 ± 2.9
9283.5 ± 0.99283.4X9255.4 ± 0.99255.4X PsbE
17934.6 ± 2.218056.5 ± 1.8e + O
17919.6 ± 1.118040.2 ± 1.6e
4409.3 ± 0.53972.1 ± 0.4
4272.7 ± 0.63866.0 ± 0.2
4367.1 ± 0.14366.0Y4365.9 ± 0.84366.0X PsbL
4759.7 ± 0.2
39481.7 ± 2.839475.9YAc-PsbD
39439.6 ± 2.539437.5XPsbD
56032.2 ± 6.756064.0Ac-PsbB
10101.1 ± 1.6
7774.0 ± 0.67774.9YPsbH + 2P + O
7792.4 ± 0.57823.0 2 7694.3 ± 0.67694.9XPsbH + P + O
7713.9 ± 0.87743.0 2 7613.9 ± 1.17614.9YPsbH + O
7857.4 ± 1.17887.0 2 7758.8 ± 0.67758.9XPsbH + 2P
7777.3 ± 0.97807.0 2 7679.3 ± 0.97678.9X PsbH + P
7697.3 ± 1.07727.0 2 7599.5 ± 1.27598.9X PsbH
5913.2 ± 0.45927.7 ± 0.45927.7XPsbW
17329.3 ± 1.617329.6XPetD + O
17359.1 ± 1.215136.0 3 17313.7 ± 2.017313.6XPetD
4140.6 ± 0.64112.5 4 fM-PsbT + P
4060.8 ± 0.74032.5 4 3849.0 ± 0.43849.7Y fM-PsbT
4284.9 ± 0.3
50205.9 ± 8.350204.9XAc-PsbC
3809.8 ± 0.63810.6Y fM-PsbM
4212.0 ± 0.64211.9XfM-PsbI + O
4210.3 ± 0.64209.8Y4195.8 ± 0.54195.9X fM-PsbI
  • a IMTs assembled in order of elution during the primary reverse-phase chromatographic elution. The mean ± S.D. of n experiments is presented.

  • b Calculated average mass of the uncharged assigned gene product shown in ID column.

  • 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, IMT 26532.1 is in the mass range and retention time for PsbO, except the mass does not match the published sequence. The mass difference of 129 Da could reasonably be expected to be because of one or more amino acid changes. Removal of a single Q from the C terminus (26533.7 calculated) results in excellent agreement, for example. 2, the pea PsbH sequence has at least two amino acid changes from the published sequence. We have identified one (A18V) by MS/MS (not shown). 3, the pea petD sequence does not include the first exon and erroneously starts with an AUG codon in the second exon. A chimeric protein translated from the first exon of the spinach sequence and the second exon from pea gives a calculated mass of 17369.7 Da. The mass difference is consistent with changing either Pro8 or Pro12 encoded in exon 1 of spinach petD to Ser. 4, the published pea psbT gene sequence appears to have an in-frame deletion that is currently being confirmed.

  • d Protein IDs in quotes indicate assignments made by retention times consistent with membrane localization and similar masses from published sequences from other plants. IDs in italic type indicate that a tobacco IMT (not shown) was correlated with a published tobacco gene sequence and that the protein eluted at a similar time to the pea and spinach orthologs. O, oxygen; P, phosphate; Ac, acetyl; F, formyl; fM, N-formylmethionine. Five unassigned post-translationally modified masses are labeled in lower case letters (a–e) in order of elution. All accession numbers are from GenBank™, unless otherwise noted. Pea: psaC, X13157; psbP, X15552; psbO, X15350; petC, X63065; psbF, psbJ, psbE, psbL, X15767; Ihcb1*4, X56338; Ihcb1*2, K02067; Ihcb2*1, X57082; Ihcb3*1, X69215; psbD, M27309; psbB, psbH, psbT, AF153442; petD, X00535; psbA, M11005; psbC, M27309; psbM, D12535; PsbI (see Ref. 39). Spinach: PsbX, pir# SO3277 (unidentified X changed to Cys); psaE, X14018; psaD, X14617; psbP, X05511; psbQ, X05512; psbO, X05548; psaH, X16858; petC, X06244; psbF, psbE, psbL, M35673; psbR, J03887; psaF, X13133; Ihcb1*1, X14341; Ihcb6*1, Z25886; psbS, X68552; psbY2, AF060198; psbB, NP054960; psbH, NP054963 amino acids 8–79 (earlier entries initiate from the second methionine 7, but internal sequence errors are corrected in this sequence); psbT, P37259; psbM, NP054926; psbI, NP054916; psbW, X85038; petD, X07106.