Laser Ablation Sample Transfer for Localized LC-MS/MS Proteomic Analysis of Tissue

F.Donnarumma and K. K. Murray, Laser ablation sample transfer for localized LC-MS/MS proteomic analysis of tissue, J Mass Spectrom, 2016 51 261-268.

Laser Ablation Sample Transfer for Localized LC-MS/MS Proteomic Analysis of Tissue. J. Mass Spectrom. 2016, 51, 261
Donnarumma, F.; Murray, K. K. Laser Ablation Sample Transfer for Localized LC-MS/MS Proteomic Analysis of Tissue. J. Mass Spectrom. 2016, 51, 261–268.

We have developed a mid-infrared laser ablation sampling technique for nano-flow liquid chromatography coupled with tandem mass spectrometry proteomic profiling of discrete regions from biological samples. Laser ablation performed in transmission geometry was used to transfer material from 50-μm thick tissue sections mounted on a glass microscope slide to a capturing solvent. Captured samples were processed using filter-aided sample preparation and enzymatically digested to produce tryptic peptides for data-dependent analysis with an ion trap mass spectrometer. Comparison with ultraviolet laser capture microdissec- tion from neighboring regions on the same tissue section revealed that infrared laser ablation transfer has higher reproducibility between samples from different consecutive sections. Both techniques allowed for proteomics investigation of different orga- nelles without the addition of surfactants.

Isolation and determination of the primary structure of a lectin protein from the serum of the american alligator (Alligator Mississippiensis)

Darville, Merchant, Maccha, Siddavarapu, Hasan, and Murray
Comp. Biochem Physiol. B

Alligator Lectin Protein Sequence (doi:10.1016/j.cbpb.2011.11.001)

Mass spectrometry in conjunction with de novo sequencing was used to determine the amino acid sequence of a 35 kDa lectin protein isolated from the serum of the American alligator that exhibits binding to mannose. The protein N-terminal sequence was determined using Edman degradation and enzymatic digestion with different proteases was used to generate peptide fragments for analysis by liquid chromatography tandem mass spectrometry (LC MS/MS). Separate analysis of the protein digests with multiple enzymes enhanced the protein sequence coverage. De novo sequencing was accomplished using MASCOT Distiller and PEAKS software and the sequences were searched against the NCBI database using MASCOT and BLAST to identify homologous peptides. MS analysis of the intact protein indicated that it is present primarily as monomer and dimer in vitro. The isolated 35 kDa protein was ~ 98% sequenced and found to have 313 amino acids and nine cysteine residues and was identified as an alligator lectin. The alligator lectin sequence was aligned with other lectin sequences using DIALIGN and ClustalW software and was found to exhibit 58% and 59% similarity to both human and mouse intelectin-1. The alligator lectin exhibited strong binding affinities toward mannan and mannose as compared to other tested carbohydrates.

A solid-phase bioreactor with continuous sample deposition for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

DOI: 10.1002/rcm.4921

Lee, Soper, and Murray
Rapid Commun. Mass Spectrom. 2011, 25, 693–699

We report the development of a solid-phase proteolytic digestion and continuous deposition microfluidic chip platform for low volume fraction collection and off-line matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Tryptic peptides were formed in an on-chip bioreactor and continuously deposited onto a MALDI target plate using a motor-driven xyz stage. The bioreactor consisted of a 4cmT200mmT50mm microfluidic channel with covalently immobilized trypsin on an array of 50mm diameter micropost structures with a 50 mm edge-to-edge inter-post spacing. A 50 mm i.d. capillary tube was directly attached to the end of the bioreactor for continuous sample deposition. The MALDI target plate was modified by spin-coating a nitrocellulose solution containing a MALDI matrix on the surface prior to effluent deposition. Protein molecular weight standards were used for evaluating the performance of the digestion and continuous deposition system. Serpentine sample traces 200 mm wide were obtained with a 30 fmol/mm quantity deposition rate and a 3.3 nL/mm volumetric deposition rate.

McNeese March 2009

In March of 2009, I visited McNeese State University in Lake Charles, Louisiana to give a talk and visit my collaborator Prof. Mark Merchant, “the alligator man”. Mark gave me a tour of his alligator holding facilities on campus.

Alligator in pen at McNeese State University, March 2009

Mark wanted to get this alligator to open its mouth so that I could get a good photo.

Prof. Mark Merchant with an alligator at McNeese State University March 2009
Prof. Mark Merchant with an alligator at McNeese State University March 2009

Sure enough, the alligator did open his mouth. Note Chemistry Department Head Ron Darbeau who was on the proper side of the fence during all of this. I was 15 feet away and the gate was open.

Prof. Mark Merchant testing alligator stimulus and response at McNeese State University March 2009.
Alligator at McNeese State University March 2009

In the next cage over, Mark scooped out a handful of baby alligators.

Prof. Mark Merchant with baby alligators at McNeese State University March 2009

I call the one on the right “Bitey” – he clamped down on my knuckle when I unwisely offered it to him.

Baby Alligators at McNeese State University March 2009