Table 1.

Labeling check for small amounts of peptides

TMT label	126	127N	127C	128N	128C	129N	129C	130N	130C	131
Amount analyzed	100 ng	100 ng	100 ng	100 ng	100 ng	100 ng	100 ng	100 ng	100 ng	100 ng
Total peptide #	4216	4045	4547	4332	4858	4121	4519	4724	5172	5204
Unmodified peptide #	227	230	201	189	238	243	239	217	255	252
N-Acetyl TMT #	75	50	61	58	80	69	84	74	84	79
N-Acetyl TMT %	1.8%	1.2%	1.3%	1.3%	1.6%	1.7%	1.9%	1.6%	1.6%	1.5%
N term TMT #	3516	3458	3937	3736	4151	3456	3861	4015	4717	4444
N term TMT %	84%	86%	87%	86%	85%	84%	85%	85%	91%	85%
Lys TMT #	2680	2635	2949	2739	3097	2561	2898	3017	3360	3411
Lys TMT %	64%	65%	65%	63%	64%	62%	64%	64%	65%	66%
Either N term or Lys TMT #	3989	3815	4346	4143	4620	3878	4280	4507	4917	4952
All TMT % peptides (%)	95%	94%	96%	96%	95%	94%	95%	95%	95%	95%

TMT label incorporation results are shown for small amounts of peptides. The labeled peptides were reconstituted in 20 μL 0.1% formic acid, desalted using Stage Tips and eluted in 20 μL of 40% acetonitrile/0.1% formic acid, dried, and reconstituted in 10 μL 0.1% formic acid. Eight microliter were analyzed by LC-MS/MS. This optimized experiment for 100 ng of quantified yeast peptide pools is used for determining sample amounts for scaled up labeling