PMID- 26247297
OWN - NLM
STAT- MEDLINE
DCOM- 20160405
LR  - 20240213
IS  - 1750-2799 (Electronic)
IS  - 1754-2189 (Print)
IS  - 1750-2799 (Linking)
VI  - 10
IP  - 9
DP  - 2015 Sep
TI  - Complementary IMAC enrichment methods for HLA-associated phosphopeptide 
      identification by mass spectrometry.
PG  - 1308-18
LID - 10.1038/nprot.2015.086 [doi]
AB  - Phosphorylation events within cancer cells often become dysregulated, leading to 
      oncogenic signaling and abnormal cell growth. Phosphopeptides derived from 
      aberrantly phosphorylated proteins that are presented on tumors and not on normal 
      tissues by human leukocyte antigen (HLA) class I molecules are promising 
      candidates for future cancer immunotherapies, because they are tumor specific and 
      have been shown to elicit cytotoxic T cell responses. Robust phosphopeptide 
      enrichments that are suitable for low input amounts must be developed to 
      characterize HLA-associated phosphopeptides from clinical samples that are 
      limited by material availability. We present two complementary mass 
      spectrometry-compatible, iron(III)-immobilized metal affinity chromatography 
      (IMAC) methods that use either nitrilotriacetic acid (NTA) or iminodiacetic acid 
      (IDA) in-house-fabricated columns. We developed these protocols to enrich for 
      subfemtomole-level phosphopeptides from cell line and human tissue samples 
      containing picograms of starting material, which is an order of magnitude less 
      material than what is commonly used. In addition, we added a peptide 
      esterification step to increase phosphopeptide specificity from these low-input 
      samples. To date, hundreds of phosphopeptides displayed on melanoma, ovarian 
      cancer, leukemia and colorectal cancer have been identified using these highly 
      selective phosphopeptide enrichment protocols in combination with a program 
      called 'CAD Neutral Loss Finder' that identifies all spectra containing the 
      characteristic neutral loss of phosphoric acid from phosphorylated serine and 
      threonine residues. This methodology enables the identification of HLA-associated 
      phosphopeptides presented by human tissue samples containing as little as 
      nanograms of peptide material in 2 d.
FAU - Abelin, Jennifer G
AU  - Abelin JG
AD  - Department of Chemistry, University of Virginia, Charlottesville, Virginia, USA.
FAU - Trantham, Paisley D
AU  - Trantham PD
AD  - Department of Chemistry, University of Virginia, Charlottesville, Virginia, USA.
FAU - Penny, Sarah A
AU  - Penny SA
AD  - Department of Clinical Immunology, University of Birmingham, Birmingham, UK.
FAU - Patterson, Andrea M
AU  - Patterson AM
AD  - Department of Microbiology and Immunology, University of Oklahoma, Oklahoma City, 
      Oklahoma, USA.
FAU - Ward, Stephen T
AU  - Ward ST
AD  - Department of Clinical Immunology, University of Birmingham, Birmingham, UK.
FAU - Hildebrand, William H
AU  - Hildebrand WH
AD  - Department of Microbiology and Immunology, University of Oklahoma, Oklahoma City, 
      Oklahoma, USA.
FAU - Cobbold, Mark
AU  - Cobbold M
AD  - Department of Clinical Immunology, University of Birmingham, Birmingham, UK.
FAU - Bai, Dina L
AU  - Bai DL
AD  - Department of Chemistry, University of Virginia, Charlottesville, Virginia, USA.
FAU - Shabanowitz, Jeffrey
AU  - Shabanowitz J
AD  - Department of Chemistry, University of Virginia, Charlottesville, Virginia, USA.
FAU - Hunt, Donald F
AU  - Hunt DF
AD  - 1] Department of Chemistry, University of Virginia, Charlottesville, Virginia, 
      USA. [2] Department of Pathology, University of Virginia, Charlottesville, 
      Virginia, USA.
LA  - eng
GR  - GM 037537/GM/NIGMS NIH HHS/United States
GR  - R37 AI033993/AI/NIAID NIH HHS/United States
GR  - R01 AI033993/AI/NIAID NIH HHS/United States
GR  - T32 AI007633/AI/NIAID NIH HHS/United States
GR  - R01 GM037537/GM/NIGMS NIH HHS/United States
GR  - AI 033993/AI/NIAID NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
DEP - 20150806
PL  - England
TA  - Nat Protoc
JT  - Nature protocols
JID - 101284307
RN  - 0 (Histocompatibility Antigens)
RN  - 0 (Phosphopeptides)
SB  - IM
MH  - Chromatography, Affinity/*methods
MH  - Histocompatibility Antigens/metabolism
MH  - Mass Spectrometry
MH  - Phosphopeptides/*analysis/metabolism
PMC - PMC4640213
MID - NIHMS731387
EDAT- 2015/08/08 06:00
MHDA- 2016/04/06 06:00
PMCR- 2016/09/01
CRDT- 2015/08/07 06:00
PHST- 2015/08/07 06:00 [entrez]
PHST- 2015/08/08 06:00 [pubmed]
PHST- 2016/04/06 06:00 [medline]
PHST- 2016/09/01 00:00 [pmc-release]
AID - nprot.2015.086 [pii]
AID - 10.1038/nprot.2015.086 [doi]
PST - ppublish
SO  - Nat Protoc. 2015 Sep;10(9):1308-18. doi: 10.1038/nprot.2015.086. Epub 2015 Aug 6.